Methods of diagnosing and treating neonatal reversion to fetal consciousness

ABSTRACT

This invention provides methods of diagnosing and treating syndromes of reversion to fetal consciousness in a neonate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/162,753, filed on Jan. 24, 2014, and issued as U.S. Pat. No.9,339,433 on May 17, 2016, which claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Application No. 61/756,957, filed on Jan.25, 2013, which are hereby incorporated herein by reference in theirentireties for all purposes.

FIELD

The present invention relates to methods of diagnosing and treatingsyndromes of reversion to fetal consciousness in a neonate.

BACKGROUND

Neonatal maladjustment syndrome (NMS) is a common disorder of neonatalfoals that manifests within the first 72 h of life (Bernard, et at(1995) In: Proceedings, 41st American Association of EquinePractitioners, Lexington, Ky. pp 222-224; Rossdale and Leadon, (1975) JReprod Fertil 23, 658-661). The proposed mechanisms include hypoxic andischaemic events prior to, during and shortly after parturition (Palmerand Rossdale, (1976) Res Vet Sci 20, 267-275). Affected foals exhibitneurological dysfunction such as seizures and altered states ofconsciousness, behaviour and response to stimuli (Bernard et al. 1995,supra; Ringger, et al. (2011) J Vet Intern Med 25, 132-137). However,hypoxic and ischaemic injury is not always identified uponhistopathological evaluation, and long-term neurological deficits havebeen reportedly rare. Fetal corticosteroids, through activation of thehypothalamo-pituitary-adrenocortical (HPA) axis, contribute to thematuration of many organs and regulate the transition between intra- andextrauterine life (Rossdale, (2004) In: Proceedings, 51st AmericanAssociation of Equine Practitioners, Denver, Colo. pp 75-126). Rossdale,et al., ((1995) Reprod Fertil Dev 7, 567-575) reported increasedconcentrations of progestagens in neonatal foals that rapidly decreaseover the following 48 h after birth (Houghton, et al., (1991) J ReprodFert Suppl 44, 609-617; Rossdale 2004, supra). Foals with NMS have beenreported to have persistently increased concentrations of plasmaprogestagens (Houghton et al. 1991, supra; Rossdale et al. 1995, supra;Rossdale 2004, supra). Concentrations of several plasma steroidsincluding progestagens (progesterone and pregnenolone) and androgens(epitestosterone and androstenedione) were found to be significantlyincreased in foals with NMS compared with healthy foals (Aleman et al,(2013) Equine Veterinary Journal). Certain steroidal compounds calledneurosteroids, predominantly 5-α reduced pregnanes, can cross theblood-brain barrier and have neuromodulatory effects (Mellon and Griffin(2002) Trends Endocrinol Metab 13, 35-43; Naert, et al. (2007)Psychoneuroendocrinology 32, 1062-1078). It is proposed that in a subsetof foals the signs of NMS may not be the result of hypoxia, and thatthese neurosteroids may play a role in the aetiology and clinicalmanifestations of foals with NMS.

NMS has been referred to as hypoxic-ischemic encephalopathy, perinatalasphyxia, neonatal encephalopathy, and dummy foal syndrome (Drummond(1988) Equine Vet J 5, 41-43; Vaala, (1994) Vet Clin N Am Equine Pract10, 187-218). The proposed pathogenesis is the result of hypoxia andischemia of the brain which occurs shortly before, during or afterparturition leading to neuronal cellular energy failure and death(Drummond (1988) Equine Vet J 5, 41-43; Rossdale (1972) Equine Vet J 4,117-128; Ringger, et al., (2011) J Vet Intern Med 25, 132-137). Clinicalsigns are consistent with brain hypoxia and include alterations in thestate of consciousness from mild obtundation to stuporous to comatose;abnormal behaviour such as lack of affinity for the mare, not nursing,vocalization, and wandering; blindness; and paroxysmal activity such aspaddling and seizures (Palmer and Rossdale (1976) Res Vet Sci 20,267-275). Histopathological evidence of cerebral hemorrhage and hypoxiahas been detected in some severely affected foals (Palmer and Rossdale(1976), supra). However, many foals do not have histological evidence ofhypoxia, edema or hemorrhage (Bernard, et al., In: Proceedings, 41stAmerican Association of Equine Practitioners, Lexington, Ky. pp222-224). Furthermore, many foals have a normal birth and recoverquickly and fully from the condition. This is in contrast to infants andnewborn rats with asphyxia in which a significantly longer recovery timeis needed and long-term neurological deficits are often manifest (Kiss,et al., (2009) Brian Res 1255, 42-50; van Handel, et al., (2007) Eur JPediatr 166, 645-654). The fast recovery with no apparent long-termdeficits and lack of evidence of hypoxia or ischemia in affectedneonatal foals suggest that the syndrome may not be exclusively theresult of hypoxia.

Neonatal foals have high concentrations of pregnanes at birth whichdecrease rapidly over the first 48 hours of life (Houghton, et al.,(1991) J Reprod Fertil 44, 609-617). Elevated concentrations of plasmapregnanes and a correlation between decreasing levels of pregnanes andclinical recovery have been reported (Rossdale, et al., (1995) ReprodFertil Dev 7, 567-575). Certain steroidal compounds, predominantly 5-αreduced pregnanes, appear to have important neuromodulatory roles(Baulieu, (1998) Psychoneuroendocrinology 23, 963-987; Mellon andGriffin (2002) Trends Endocrinol Metab 13, 35-43; Robel and Baulieu,(1994) Trends Endocrinol Metab 5, 1-8). These steroids are synthesizedde novo in glial cells from cholesterol or blood-borne steroidprecursors (Robel, et al., (1994) Trends Endocrinol Metab 5:1-8) and arepotent allosteric modulators of the GABA_(A) receptor; lowconcentrations cause weak enhancement of GABA activity and highconcentrations cause complete non-competitive inhibition (Baulieu (1998)Psychoneuroendocrinology 23, 963-987). Infusion of certain 5α-reducedpregnanes into rats and mice (Naert, et al., (2007)Psychoneuroendocrinology 32, 1062-1078; Zhu, et al., (2001) Br J Anaesth86, 403-412) and neonatal foals (Madigan, et al., (2012) Equine Vet J 44S41 109-112.) leads to anaesthesia or marked behavioural effectssuggesting that these pregnanes cross the blood brain barrier and exertneuromodulatory effects.

SUMMARY

In one aspect, provided are methods of diagnosing a syndrome ofreversion to fetal consciousness in a neonate mammal. In someembodiments, the methods comprise:

a) measuring or assaying the levels of one or more pregnanes in abiological sample from the neonate mammal and;

b) diagnosing the neonate mammal as suffering reversion to fetalconsciousness when elevated or detectable levels of the one or morepregnanes are measured.

In a further aspect, provided are methods of diagnosing a syndrome ofreversion to fetal consciousness in a neonate mammal. In someembodiments, the methods comprise:

a) receiving a report providing the levels of one or more pregnanes in abiological sample from the neonate mammal and;

b) diagnosing the neonate mammal as suffering reversion to fetalconsciousness when elevated or detectable levels of the one or morepregnanes are measured.

In another aspect, provided are methods of diagnosing a syndrome ofreversion to fetal consciousness in a neonate mammal. In someembodiments, the methods comprise:

a) measuring or assaying a level of one or more neurosteroids in abiological sample from the neonate mammal and;

b) diagnosing the neonate mammal as suffering reversion to fetalconsciousness when the level of the one or more neurosteroids iselevated above a threshold level.

In a further aspect, provided are methods of diagnosing a syndrome ofreversion to fetal consciousness in a neonate mammal. In someembodiments, the methods comprise:

a) receiving a report providing a level of one or more neurosteroids ina biological sample from the neonate mammal and;

b) diagnosing the neonate mammal as suffering reversion to fetalconsciousness when the level of the one or more neurosteroids iselevated above a threshold level.

In a further aspect, provided are methods of detecting an indicator ofreversion to fetal consciousness in a neonatal mammalian subject, themethod comprising assaying a blood sample from the subject for a panelof neurosteroids, wherein the presence of one or more of theneurosteroids at a detectable level or a level above a predeterminedthreshold level indicates the presence reversion to fetal consciousnessin the subject.

With respect to embodiments of the diagnostic methods, in someembodiments, the neonate mammal is selected from the group consisting ofa human, a non-human primate, Equidae, Bovidae, Cervidae, Suidae,Canidae, Felidae, Rodentia, Lagomorpha, Camelidae, Ursidae, Procyonidae,Mustelidae, Elephantidae. In some embodiments, the neonate mammal is anequine. In some embodiments, the neonate mammal is exhibiting symptomsof reversion to fetal consciousness (e.g., diminished mentation,inability and/or unwillingness to breathe properly using lungs,inability and/or unwillingness to nurse, inability to stand and/orunstable gait).

In varying embodiments, the one or more neurosteroids measured orassayed comprise one or more pregnanes, one or more androgens and/or oneor more estrogens. In varying embodiments, the panel of neurosteroidsassayed or measured comprises one or more or all pregnanes selected fromthe group consisting of allopregnanolone (3α-hydroxy-5α-pregnan-20-oneor 3α,5α-tetrahydroprogesterone; also abbreviated as THP or THPROG),allopregnanolone sulfate, 5α-dihydroprogesterone (DHP),17-hydroxypregnenolone, 17-hydroxyprogesterone, pregnanediol,pregnenolone, pregnenolone sulfate and progesterone; one or more or allandrogens/estrogens selected from the group consisting ofandrostenedione, 1,4-androstadiene-3,17-1, boldenone, boldenone sulfate,dehydroepiandrosterone (DHEA), 5-α dihydroandrolone, 5-αdihydrotestosterone, 5-β dihydrotestosterone, epinandrolone,epistestosterone, 17-α estradiol, 17-β estradiol, 17-β estradiolsulfate, 5-α-estran-3-β-17 diol, estrone, estrone sulfate,6-α-hydroxyandrostenedione, nandrolone, nandrolone glucuronide,nandrolone sulfate, 19-norandrostenedione, 19-nor-androsterone,19-norepiandrosterone, testosterone, testosterone glucuronide andtestosterone sulfate. Additionally, one or more or all compoundsselected from the group consisting of adrenocorticotropic hormone(ACTH), adenosine, cortisol and oxytocin may be assayed or measured. Invarying embodiments, the pregnanes measured comprise neurosteroidalactivity. In varying embodiments, the pregnanes measured compriseadrenally-derived pregnanes. In varying embodiments, the level of one ormore, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9, pregnanes selected from thegroup consisting of allopregnanolone (3α-hydroxy-5α-pregnan-20-one or3α,5α-tetrahydroprogesterone; also abbreviated as THP or THPROG),allopregnanolone sulfate, 5α-dihydroprogesterone (DHP),17-hydroxypregnenolone, 17-hydroxyprogesterone, pregnanediol,pregnenolone, pregnenolone sulfate and progesterone is measured orassayed. In some embodiments, one or more, e.g., one, two, three or fourpregnanes, selected from the group consisting of progesterone,pregnenolone, 17OH progesterone and pregnanediol are measured. In someembodiments, one or more pregnanes selected from the group consisting ofprogesterone and pregnenolone are measured. In varying embodiments, thelevels of pregnenolone in the biological sample are measured. The levelsof the one or more pregnanes in the biological sample can be comparedwith a predetermined threshold level. In some embodiments, the level ofone or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27, androgens/estrogensselected from the group consisting of androstenedione,1,4-androstadiene-3,17-1, boldenone, boldenone sulfate,dehydroepiandrosterone (DHEA), 5-α dihydroandrolone, 5-αdihydrotestosterone, 5-β dihydrotestosterone, epinandrolone,epistestosterone, 17-α estradiol, 17-β estradiol, 17-β estradiolsulfate, 5-α-estran-3-β-17 diol, estrone, estrone sulfate,6-α-hydroxyandrostenedione, nandrolone, nandrolone glucuronide,nandrolone sulfate, 19-norandrostenedione, 19-nor-androsterone,19-norepiandrosterone, testosterone, testosterone glucuronide andtestosterone sulfate is assayed or measured. In varying embodiments, thelevel of one or more compounds selected from the group consisting ofadrenocorticotropic hormone (ACTH), adenosine, cortisol and oxytocin isadditionally assayed or measured. Levels of the one or moreneurosteroids, e.g., the one or more pregnanes and/or the one or moreandrogens/estrogens above the predetermined threshold level indicateand/or are associated with a positive diagnosis of reversion to fetalconsciousness. In varying embodiments, the predetermined threshold levelfor the neurosteroids selected from allopregnanolone(3α-hydroxy-5α-pregnan-20-one or 3α,5α-tetrahydroprogesterone; alsoabbreviated as THP or THPROG), allopregnanolone sulfate,5α-dihydroprogesterone (DHP), 17-hydroxypregnenolone,17-hydroxyprogesterone, pregnanediol, pregnenolone sulfate,1,4-androstadiene-3,17-1, boldenone, boldenone sulfate, 5-αdihydroandrolone, 5-α dihydrotestosterone, 5-β dihydrotestosterone,epinandrolone, 17-α estradiol, 17β estradiol, 17-β estradiol sulfate,5-α-estran-3-β-17 diol, estrone, estrone sulfate,6-α-hydroxyandrostenedione, nandrolone, nandrolone glucuronide,nandrolone sulfate, 19-norandrostenedione, 19-nor-androsterone,19-norepiandrosterone, testosterone, testosterone glucuronide andtestosterone sulfate, adrenocorticotropic hormone (ACTH), adenosine,cortisol and oxytocin is above 0.0001 ng/mL or any detectable level. Invarying embodiments, the predetermined threshold level for androsterone,DHEA, epitestosterone, progesterone and pregnenolone at ages 0-few hoursof age, 24 h, and 48 h old are as shown in Table 1. In varyingembodiments, the threshold value for ACTH is 53 ng/mL), the thresholdvalue for cortisol is 16 ng/mL, the threshold value for adenosine is 0.3ng/mL), and the threshold value for oxytocin is 1.1 ng/mL.

TABLE 1 Steroid (ng/mL) 0 h 24 h 48 h androstenedione 0.6 0.3 0.0001(any detectable level) DHEA 50 25 8 epitestosterone 0.2 0.05 0.004progesterone 4 0.0001 (any 0.0001 (any detectable level) detectablelevel) pregnenolone 1200 193 104

In varying embodiments, the elevated pregnenolone levels are above about6000 ng/ml under 24 hours post birth, above about 550 ng/ml at 24-28hours post birth and/or above about 315 ng/ml at or after 48 hours postbirth. In varying embodiments, the elevated pregnenolone levels areabove about 12,900 ng/ml under 24 hours post birth, above about 3470ng/ml at 24-28 hours post birth and/or above about 3420 ng/ml at orafter 48 hours post birth. In varying embodiments, the elevatedprogesterone levels are above about 10.5 ng/ml under 24 hours postbirth, above about 0.8 ng/ml at 24-28 hours post birth and/or aboveabout 0.1 ng/ml at or after 48 hours post birth. In varying embodiments,the elevated progesterone levels are above about 9.7 ng/ml at 24-28hours post birth and/or above about 14.3 ng/ml at or after 48 hours postbirth.

In varying embodiments, the diagnostic methods further comprisemeasuring the level of one or more androgens/estrogens (in addition tothe level of one or more pregnanes) in the biological sample. In someembodiments, the one or more androgens are selected from the groupconsisting of androstenedione, dehydroepiandrosterone (DHEA),DHEA-sulphate and epitestosterone are measured. In some embodiments, thelevel of one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27,androgens/estrogens selected from the group consisting ofandrostenedione, 1,4-androstadiene-3,17-1, boldenone, boldenone sulfate,dehydroepiandrosterone (DHEA), 5-α dihydroandrolone, 5-αdihydrotestosterone, 5-β dihydrotestosterone, epinandrolone,epistestosterone, 17-α estradiol, 17-β estradiol, 17-β estradiolsulfate, 5-α-estran-3-β-17 diol, estrone, estrone sulfate,6-α-hydroxyandrostenedione, nandrolone, nandrolone glucuronide,nandrolone sulfate, 19-norandrostenedione, 19-nor-androsterone,19-norepiandrosterone, testosterone, testosterone glucuronide andtestosterone sulfate is assayed or measured.

In varying embodiments, the biological sample is a fluid sample, e.g.,selected from the group consisting of blood, serum, plasma, urine andsaliva. In varying embodiments, the neonate is less than 120 hours postbirth, e.g., less than 96, 84, 72, 60, 48, 36, 24, 12, 9, 6, 3, 2, 1hours post birth. In varying embodiments, the neurosteroid (e.g.,pregnane) levels are measured at two or more time points, e.g., two,three, four, five or more time points. A diagnosis of reversion to fetalconsciousness is indicated when the levels of neurosteroids (e.g.,pregnanes) remain elevated, increase or do not decrease at subsequenttime points.

In varying embodiments, the diagnostic methods further comprise, uponmeasuring elevated neurosteroid (e.g., pregnane) levels and positivelydiagnosing the neonate mammal as suffering reversion to fetalconsciousness, administering to the neonate an effective amount of a 5αreductase inhibitor. In some embodiments, the 5α reductase inhibitor isselected from the group consisting of alfatradiol, dutasteride,finasteride, bexlosteride, epristeride, izonsteride, lapisteride,turosteride, and analogs, salts and mixtures thereof. In someembodiments, the 5α reductase inhibitor is dutasteride.

In varying embodiments, the diagnostic methods further comprise, uponmeasuring elevated neurosteroid (e.g., pregnane) levels and positivelydiagnosing the neonate mammal as suffering reversion to fetalconsciousness, subjecting the neonate to squeezing or hugging along themid-thorax. In varying embodiments, the applied pressure of thesqueezing is maintained for at least 10 minutes, e.g., at least 15, 20,25, 30, 35, 40, 45, 50, 55 or 60 minutes. In varying embodiments, thesqueezing or hugging of the neonate applies substantially uniformpressure along the mid-thorax of the neonate. In varying embodiments,the squeezing or hugging of the neonate comprises skin-to-skin contact.

In a further aspect, methods of preventing, reversing and/or mitigatinga syndrome of reversion to fetal consciousness in a neonate mammal. Insome embodiments, the methods comprise administering to the neonate aneffective amount of a 5α reductase inhibitor. In varying embodiments,the 5α reductase inhibitor is selected from the group consisting ofalfatradiol, dutasteride, finasteride, bexlosteride, epristeride,izonsteride, lapisteride, turosteride, and analogs, salts and mixturesthereof. In varying embodiments, the 5α reductase inhibitor isadministered to the neonate less than 120 hours post birth, e.g., lessthan 96, 84, 72, 60, 48, 36, 24, 12, 9, 6, 3, 2, 1 hours post birth.

In another aspect, methods of preventing, reversing and/or mitigating asyndrome of reversion to fetal consciousness in a neonate mammal. Insome embodiments, the methods comprise subjecting the neonate tosqueezing or hugging along the mid-thorax. In varying embodiments, theapplied pressure of the squeezing is maintained for at least 10 minutes,e.g., at least 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. Invarying embodiments, the squeezing or hugging of the neonate appliessubstantially uniform pressure along the mid-thorax of the neonate. Invarying embodiments, the squeezing or hugging of the neonate comprisesskin-to-skin contact.

With respect to embodiments of the methods for preventing, reversingand/or mitigating a syndrome of reversion to fetal consciousness in aneonate mammal, in varying embodiments, the neonate is less than 120hours post birth, e.g., less than 96, 84, 72, 60, 48, 36, 24, 12, 9, 6,3, 2, 1 hours post birth. In varying embodiments, the neonate mammal haselevated levels of neurosteroids (e.g., pregnanes) in a fluid biologicalsample, e.g., in a blood, serum, plasma, urine and/or saliva sample. Insome embodiments, one or more, e.g., one, two, three or four pregnanesselected from the group consisting of progesterone, pregnenolone, 17OHprogesterone and pregnanediol are elevated. In some embodiments, one orboth pregnanes selected from the group consisting of progesterone andpregnenolone are elevated. In some embodiments, pregnenolone iselevated. The levels of the one or more neurosteroids (e.g., pregnanes)in the biological sample can be compared with a predetermined thresholdlevel.

In varying embodiments, the elevated pregnenolone levels are above about6000 ng/ml under 24 hours post birth, above about 550 ng/ml at 24-28hours post birth and/or above about 315 ng/ml at or after 48 hours postbirth. In varying embodiments, the elevated pregnenolone levels areabove about 12,900 ng/ml under 24 hours post birth, above about 3470ng/ml at 24-28 hours post birth and/or above about 3420 ng/ml at orafter 48 hours post birth. In varying embodiments, the elevatedprogesterone levels are above about 10.5 ng/ml under 24 hours postbirth, above about 0.8 ng/ml at 24-28 hours post birth and/or aboveabout 0.1 ng/ml at or after 48 hours post birth. In varying embodiments,the elevated progesterone levels are above about 9.7 ng/ml at 24-28hours post birth and/or above about 14.3 ng/ml at or after 48 hours postbirth. In varying embodiments, the levels of one or more androgens inthe biological sample are elevated. In some embodiments, the one or moreelevated androgens are selected from the group consisting ofandrostenedione, dehydroepiandrosterone (DHEA), DHEA-sulphate andepitestosterone. In some embodiments, the neonate mammal is selectedfrom the group consisting of a human, a non-human primate, Equidae,Bovidae, Cervidae, Suidae, Canidae, Felidae, Rodentia, Lagomorpha,Camelidae, Ursidae, Procyonidae, Mustelidae, Elephantidae. In someembodiments, the neonate mammal is an equine.

In a further aspect, an inflatable cuff for squeezing and/or hugging themid-thorax of a neonate mammal is provided. In some embodiments, theinflatable cuff comprises a pneumatic inflating sleeve of sufficientcircumference to encircle the mid-thorax of a neonate mammal. In someembodiments, the pneumatic inflating sleeve is of a sufficientcircumference to encircle the mid-thorax of a neonate from a speciesselected from the group consisting of human, non-human primate, Equidae,Bovidae, Cervidae, Suidae, Canidae, Felidae, Rodentia, Lagomorpha,Camelidae, Ursidae, Procyonidae, Mustelidae, Elephantidae. In someembodiments, the neonate mammal is an equine. In some embodiments, thepneumatic inflating sleeve is of a sufficient circumference to encirclethe mid-thorax of an equine neonate. In some embodiments, the sleeve ofthe inflated cuff applies substantially even pressure along the lengthof the mid-throrax encircled by the sleeve. In some embodiments, thecuff further comprises straps for stably positioning the cuff around themid-thorax. In some embodiments, the straps of the cuff are configuredto encircle the neck of the mammal. In some embodiments, the cuff is influid communication with a foot pump or motorized pump capable of andsuitable for inflating the cuff. In varying embodiments, the devicecomprises first and second inflatable cuffs.

DEFINITIONS

The term “reversion to fetal consciousness” refers to a syndromeoccurring in a mammalian neonate within the first 72 hours of lifemanifesting clinical symptoms consistent with brain hypoxia includingwithout limitation alterations in the state of consciousness from mildobtundation to stuporous to comatose; abnormal behavior such as lack ofaffinity for the mother, not nursing, blindness, vocalization, wanderingand paroxysmal activity such as paddling and seizures.

The term “neonatal maladjustment syndrome (NMS)” refers to a conditionaffecting equine foals within the first 72 hours of life. Clinical signsare consistent with brain hypoxia and include without limitationalterations in the state of consciousness from mild obtundation tostuporous to comatose; abnormal behavior including lack of affinity forthe mare, not nursing, vocalization, and wandering; blindness; andparoxysmal activity such as paddling and seizures. NMS is described andreviewed in, e.g., Hess-Dudan, et al., Equine Vet Educ (1996) 8(1):24-32and Hess-Dudan, et al., Equine Vet Educ (1996) 8(2):79-83.

The terms “neurosteroid” and “neuroactive steroid” interchangeably referto steroid compounds having the capability to alter neuronalexcitability through interaction with neurotransmitter-gated ionchannels. Illustrative neurosteroids include without limitationallopregnanolone, dehydroepiandrosterone, dehydroepiandrosteronesulfate, 5α-dihydroprogesterone, pregnenolone, progesterone andtetrahydrodeoxycorticosterone.

The term “pregnane” refers to in vivo steroid compounds stemming orderived from 5α-pregnane (a.k.a., allopregnane) and 5β-pregnane(17β-ethyletiocholane). Structurally, pregnanes have a 21 carbon nucleusand a core ring structure comprised of cyclopentanoperhydrophenanthrene.Functionally, many pregnanes have neurosteroidal activity (e.g., alterneuronal excitability through interaction with neurotransmitter-gatedion channels). Illustrative pregnanes measured in the present methodsinclude without limitation, e.g., progesterone (pregn-4-ene-3,20-dione,CAS number 57-83-0), pregnenolone (β-hydroxypregn-5-en-20-one; CASnumber 145-13-1), 17OH progesterone (17-Hydroxypregn-4-ene-3,20-dione;CAS number 68-96-2) and pregnanediol (5β-pregnane-3α,20α-diol; CASnumber 80-92-2).

The term “threshold level” refers to representative or predeterminedlevels of one or more measured compounds (e.g., neurosteroid, pregnane,androgen) in a biological sample of a neonate. The threshold level canrepresent compound levels detected in a sample from a normal control ora population of normal controls, e.g., from a neonate or population ofneonates of the same species subject to testing known to not besuffering from reversion to fetal consciousness, e.g., NMS. Thethreshold level can be determined from an individual or from apopulation of individuals. In the present diagnostic methods, levels ofone or more compounds (e.g., neurosteroid, pregnane, androgen) above thethreshold level is indicative and/or associated with reversion to fetalconsciousness, e.g., NMS.

The terms “increased levels” or “elevated levels” are generally madewith reference to a predetermined threshold level or a level of the oneor more compounds (e.g., neurosteroid, pregnane, androgen, estrogen) ina biological sample of a neonate. An increased or elevated level isdetermined when the level of one or more compounds being measured in thetest biological sample is at least about 10%, 25%, 50%, 75%, 100% (i.e.,1-fold), 2-fold, 3-fold, 4-fold or greater, in comparison to thepredetermined threshold level of same one or more compounds (e.g.,neurosteroid, pregnane, androgen, estrogen) in the same biologicalsample tissue type (e.g., blood or urine) obtained from the same species(e.g., an equine). In determining increased levels of one or morecompounds from a biological sample, usually the same tissue types arecompared.

The terms “subject,” “individual,” and “patient” interchangeably referto any mammal, as described herein.

The term “biological sample” refers to a fluid sample from a neonatemammal, e.g., blood, serum, plasma, mucous, saliva, and/or urine.

As used herein, “administering” refers to local and systemicadministration, e.g., including enteral, parenteral, pulmonary, andtopical/transdermal administration. Routes of administration forcompounds (e.g., one or more 5α reductase inhibitors) that find use inthe methods described herein include, e.g., oral (per os (P.O.))administration, nasal or inhalation administration, administration as asuppository, topical contact, transdermal delivery (e.g., via atransdermal patch), intrathecal (IT) administration, intravenous (“iv”)administration, intraperitoneal (“ip”) administration, intramuscular(“im”) administration, or subcutaneous (“sc”) administration, or theimplantation of a slow-release device e.g., a mini-osmotic pump, a depotformulation, etc., to a subject. Administration can be by any routeincluding parenteral and transmucosal (e.g., oral, nasal, vaginal,rectal, or transdermal). Parenteral administration includes, e.g.,intravenous, intramuscular, intra-arterial, intradermal, subcutaneous,intraperitoneal, intraventricular, ionophoretic and intracranial. Othermodes of delivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

The terms “systemic administration” and “systemically administered”refer to a method of administering a compound or composition to a mammalso that the compound or composition is delivered to sites in the body,including the targeted site of pharmaceutical action, via thecirculatory system. Systemic administration includes, but is not limitedto, oral, intranasal, rectal and parenteral (e.g., other than throughthe alimentary tract, such as intramuscular, intravenous,intra-arterial, transdermal and subcutaneous) administration.

The term “co-administering” or “concurrent administration”, when used,for example with respect to the compounds (e.g., one or more 5αreductase inhibitor) and/or analogs thereof and another active agent,refers to administration of the compound and/or analogs and the activeagent such that both are in the blood at the same time.Co-administration can be concurrent or sequential.

The term “effective amount” or “pharmaceutically effective amount” referto the amount and/or dosage, and/or dosage regimen of one or morecompounds necessary to bring about the desired result e.g., an amountsufficient to mitigating in a mammal one or more symptoms or behaviorsassociated with reversion to fetal consciousness, e.g., NMS, or anamount sufficient to lessen the severity of symptoms or behaviorsassociated with reversion to fetal consciousness, e.g., NMS, an amountsufficient to reduce the risk or prevent the occurrence of symptoms orbehaviors associated with reversion to fetal consciousness, e.g., NMS ina mammal (e.g., prophylactically effective amounts).

The phrase “cause to be administered” refers to the actions taken by amedical professional (e.g., a physician), or a person controllingmedical care of a subject, that control and/or permit the administrationof the agent(s)/compound(s) at issue to the subject. Causing to beadministered can involve diagnosis and/or determination of anappropriate therapeutic or prophylactic regimen, and/or prescribingparticular agent(s)/compounds for a subject. Such prescribing caninclude, for example, drafting a prescription form, annotating a medicalrecord, and the like.

As used herein, the terms “treating” and “treatment” refer to delayingthe onset of, retarding or reversing the progress of, reducing theseverity of, or alleviating or preventing either the disease orcondition to which the term applies (e.g., reversion to fetalconsciousness, e.g., NMS), or one or more symptoms of such disease orcondition.

The term “mitigating” refers to reduction or elimination of one or moresymptoms of that pathology or disease, and/or a reduction in the rate ordelay of onset or severity of one or more symptoms of that pathology ordisease, and/or the prevention of that pathology or disease (e.g.,reversion to fetal consciousness, e.g., NMS).

As used herein, the phrase “consisting essentially of” refers to thegenera or species of active pharmaceutical agents recited in a method orcomposition, and further can include other agents that, on their own donot have substantial activity for the recited indication or purpose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a diagram of progestagens.

FIGS. 2A-E illustrate median plasma steroid concentrations (ng/ml) of a)androstenedione, b) dehydroepiandrosterone (DHEA), c) epitestosterone,d) progesterone and e) pregnenolone for healthy, sick control andneonatal maladjustment syndrome (NMS) foals during the first 48 h oflife.

FIG. 3 illustrates features of an embodiment of an inflatable cuffuseful for squeezing and imparting deep touch pressure to a neonate.

FIG. 4 illustrates an embodiment of tandem inflatable cuffs on a neonateequine. The cuffs are held in place with a strap (1) around theneonate's neck. An inflatable heart-girth cuff (2) and an inflatablecaudal cuff (3) are depicted.

FIG. 5 illustrates an embodiment of tandem inflatable cuffs on a neonateequine. The cuffs are held in place with a strap (1) around theneonate's neck. An inflatable heart-girth cuff (2) and an inflatablecaudal cuff (3) are depicted. The deep touch pressure of the inflatedcuffs induces somnolence in the neonate.

DETAILED DESCRIPTION 1. Introduction

The present invention is base, in part, on the discovery that NMS iscomprised of more than one phenotype; i) foals with hypoxia and ischemiaand ii) foals with persistence of fetal hypothalamic pituitaryadrenocortical (HPA) axis and elevated pregnane (e.g., pregnenolone,progesterone and metabolites) concentrations which can recover rapidlywith no apparent residual neurological deficits. Studies presentedherein determine the steroid profile of foals with NMS and compare itwith that of foals with other neonatal diseases and healthy controlfoals.

The present invention is based, in part, on the discovery, thatneurosteroid (e.g., pregnane) concentrations of ill, neonatal foalsremain elevated following birth, reflective of a delayed, orinterrupted, transition from intra- to extra-uterine life. Accordingly,one or more progesterone and pregnenolone measurements following birthis useful in aiding diagnosis of NMS.

2. Subjects Who May Benefit

The methods of diagnosis and treatment of a syndrome of reversion tofetal consciousness (e.g., equine neonatal maladjustment syndrome) finduse to diagnose prevent, reduce, mitigate and/or treat reversion tofetal consciousness in any mammal, including humans and non-humanmammals. Illustrative non-human mammals who can benefit from the presentdiagnostic and treatment methods include, e.g., Equidae (e.g., horse,ass, zebra), Bovidae (e.g., cattle, bison, sheep, goat, yak, impala,antelope, hartebeest, wildebeest, gnu, gazelle, water buffalo, duiker),Cervidae (e.g., deer, elk, moose, reindeer, pudu, bororo, brocket,guemal, muntjac), Suidae (e.g., pig, hog, boar), Canidae (domesticateddog, wolf, fox, coyote, jackel), Felidae (e.g., domesticated cat,cheetah, ocelot, lynx, bobcat, mountain lion, leopard, puma, lion,jaguar, tiger), Rodentia (e.g., mouse, rat, guinea pig, chinchilla,agouti, porcupine, beaver, gopher), Lagomorpha (e.g., rabbit,jackrabbit, hare, pika), Camelidae (e.g., camel, llama, alpaca, guanaco,vicugna), Ursidae (e.g., bear, panda), Procyonidae (e.g., raccoon,coati, olingo), Mustelidae (polecat, weasel, ferret, mink, fisher,badger, otter, wolverine, marten, sable, ermine), Elephantidae (e.g.,elephant), rhinoceros, hippopotamus and non-human primates (e.g.,chimpanzee, bonobo, macaque, ape).

The neonate may or may not be exhibiting symptoms of reversion to fetalconsciousness or NMS (e.g., diminished mentation, inability and/orunwillingness to breathe properly using lungs, inability and/orunwillingness to nurse, poor affinity with dam, inability to standand/or unstable gait). In varying embodiments, the neonate is less than120 hours post birth, e.g., less than 96, 84, 72, 60, 48, 36, 24, 12, 9,6, 3, 2, 1 hours post birth.

3. Methods of Diagnosis

a. Measuring Levels of One or More Neurosteroids (e.g., Pregnanes) in aBiological Sample

In varying embodiments of the diagnostic methods, the levels of one ormore neurosteroids (e.g., pregnanes) in a biological sample from aneonate are measured.

In varying embodiments, the one or more neurosteroids measured orassayed comprise one or more pregnanes, one or more androgens and/or oneor more estrogens. In varying embodiments, the panel of neurosteroidsassayed or measured comprises one or more or all pregnanes selected fromthe group consisting of allopregnanolone (3α-hydroxy-5α-pregnan-20-oneor 3α,5α-tetrahydroprogesterone; also abbreviated as THP or THPROG),allopregnanolone sulfate, 5α-dihydroprogesterone (DHP),17-hydroxypregnenolone, 17-hydroxyprogesterone, pregnanediol,pregnenolone, pregnenolone sulfate and progesterone; one or more or allandrogens/estrogens selected from the group consisting ofandrostenedione, 1,4-androstadiene-3,17-1, boldenone, boldenone sulfate,dehydroepiandrosterone (DHEA), 5-α dihydroandrolone, 5-αdihydrotestosterone, 5-β dihydrotestosterone, epinandrolone,epistestosterone, 17-α estradiol, 17β estradiol, 17-β estradiol sulfate,5-α-estran-3-β-17 diol, estrone, estrone sulfate,6-α-hydroxyandrostenedione, nandrolone, nandrolone glucuronide,nandrolone sulfate, 19-norandrostenedione, 19-nor-androsterone,19-norepiandrosterone, testosterone, testosterone glucuronide andtestosterone sulfate. Additionally, one or more or all compoundsselected from the group consisting of adrenocorticotropic hormone(ACTH), adenosine, cortisol and oxytocin may be assayed or measured.

As discussed above, the one or more pregnanes measured generally have a21-carbon nucleus and may have neurosteroidal activity. In varyingembodiments, the pregnanes measured comprise adrenally-derivedpregnanes. In some embodiments, one or more, e.g., one, two, three orfour pregnanes selected from the group consisting of progesterone,pregnenolone, 17OH progesterone and pregnanediol are measured. In someembodiments, one or more pregnanes selected from the group consisting ofprogesterone and pregnenolone are measured. In varying embodiments, thelevels of pregnenolone in the biological sample are measured. In varyingembodiments, the level of one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or9, pregnanes selected from the group consisting of allopregnanolone(3α-hydroxy-5α-pregnan-20-one or 3α,5α-tetrahydroprogesterone; alsoabbreviated as THP or THPROG), allopregnanolone sulfate,5α-dihydroprogesterone (DHP), 17-hydroxypregnenolone,17-hydroxyprogesterone, pregnanediol, pregnenolone, pregnenolone sulfateand progesterone is measured or assayed. In some embodiments, one ormore, e.g., one, two, three or four pregnanes, selected from the groupconsisting of progesterone, pregnenolone, 17OH progesterone andpregnanediol are measured. In some embodiments, one or more pregnanesselected from the group consisting of progesterone and pregnenolone aremeasured. In varying embodiments, the levels of pregnenolone in thebiological sample are measured. The levels of the one or more pregnanesin the biological sample can be compared with a predetermined thresholdlevel.

In some embodiments, the level of one or more, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26 or 27, androgens/estrogens selected from the group consisting ofandrostenedione, 1,4-androstadiene-3,17-1, boldenone, boldenone sulfate,dehydroepiandrosterone (DHEA), 5-α dihydroandrolone, 5-αdihydrotestosterone, 5-β dihydrotestosterone, epinandrolone,epistestosterone, 17-α estradiol, 17β estradiol, 17β estradiol sulfate,5-α-estran-3-β-17 diol, estrone, estrone sulfate,6-α-hydroxyandrostenedione, nandrolone, nandrolone glucuronide,nandrolone sulfate, 19-norandrostenedione, 19-nor-androsterone,19-norepiandrosterone, testosterone, testosterone glucuronide andtestosterone sulfate is assayed or measured.

In varying embodiments, the level of one or more compounds selected fromthe group consisting of adrenocorticotropic hormone (ACTH), adenosine,cortisol and oxytocin is additionally assayed or measured.

In varying embodiments, the biological sample is a fluid sample, e.g.,blood, serum, plasma, urine and/or saliva. In some embodiments, thefluid sample is blood. In some embodiments, the fluid sample is urine.

In varying embodiments, the neonate is less than 120 hours post birth,e.g., less than 96, 84, 72, 60, 48, 36, 24, 12, 9, 6, 3, 2, 1 or fewerhours post birth.

In varying embodiments, the neurosteroid (e.g., pregnane) levels aremeasured at two or more time points, e.g., two, three, four, five ormore time points. For example, two or more measurements of neurosteroid(e.g., pregnane) levels may be performed, e.g., every 4, 6, 8, 10, 12,14, 16, 18, 20, 24 or 48 hours, as appropriate.

The one or more neurosteroid (e.g., pregnane) compounds can be detectedusing any method known in the art. In varying embodiments, the one ormore neurosteroid (e.g., pregnane) compounds are detected one or moreknown techniques, including, e.g., mass spectrometry (e.g., liquidchromatography mass spectrometry (LC-MS), gas chromatography massspectrometry (GC-MS), tandem mass spectrometry (MS-MS), matrix-assistedlaser desorption/ionization (MALDI), etc.), nuclear magnetic resonance(NMR), high performance liquid chromatography (HPLC), ultra-performanceliquid chromatography (UPLC), atmospheric-pressure chemical ionization(APCI) or immunoassay (e.g., enzyme-linked immunoassay (ELISA),radioimmunoassay RIA), lateral flow immunoassay, etc.). See, e.g.,Keski-Rahkonen, et al., J Steroid Biochem Mol Biol. (2011)127(3-5):396-404; Rauh, J Steroid Biochem Mol Biol. (2010)121(3-5):520-7; Kushnir, et al., Clin Biochem. (2011) 44(1):77-88;Kulle, et al., J Endocrinol Invest. (2011) 34(9):702-8; Tripathy, etal., Clin Chim Acta. (2012) 413(1-2):262-8; and Posthuma-Trumpie, etal., Anal Bioanal Chem. (2008) 392(6):1215-23.

b. Comparing the Measured Levels of Neurosteroids (e.g., Pregnanes) to aThreshold Level

Levels of the one or more neurosteroids, e.g., the one or more pregnanesand/or the one or more androgens/estrogens above the predeterminedthreshold level indicate and/or are associated with a positive diagnosisof reversion to fetal consciousness. The increased or elevated levels ofone or more measured neurosteroids is indicated by a detectable signal.In varying embodiments, the levels of the one or more measuredneurosteroid (e.g., pregnane) compounds in a test sample can be comparedto the levels of the one or more measured neurosteroid (e.g., pregnane)compounds from a negative or normal control sample or to a thresholdvalue. In some embodiments, increased levels of one or moreneurosteroids (e.g., pregnanes) are detected, and a diagnosis ofreversion to fetal conscious, e.g., NMS, is indicated, e.g., when theneonate is exhibiting symptoms and/or the test sample has one or moreneurosteroid (e.g., pregnane) compounds at levels elevated at leastabout 10%, 20%, 30%, 50%, 75% greater in comparison to the levels of thesame neurosteroid (e.g., pregnane) compounds in the normal controlsample or the predetermined threshold value. In some embodiments,increased levels of one or more neurosteroids (e.g., pregnanes) aredetected, and a diagnosis of reversion to fetal conscious, e.g., NMS, isindicated, e.g., when the neonate is exhibiting symptoms and/or the testsample has one or more neurosteroid (e.g., pregnane) compounds at levelselevated at least about 1-fold, 2-fold, 3-fold, 4 fold or more, greaterin comparison to the levels of the same neurosteroid (e.g., pregnane)compounds in the normal control sample or the predetermined thresholdvalue. Usually, the sample and control or predetermined threshold levelsare from the same tissue types.

In some embodiments, the levels of the one or more neurosteroid (e.g.,pregnane) compounds in a test sample are compared with the levels of theone or more measured neurosteroid (e.g., pregnane) compounds from apositive control biological sample of a neonate known to suffer fromreversion to fetal consciousness, e.g., NMS. In this case, levels of theone or more measured neurosteroid (e.g., pregnane) compounds in the testbiological sample equivalent to or greater than the positive controlsample, from a neonate known to suffer from reversion to fetalconsciousness, e.g., NMS, are indicative of and/or associated with apositive diagnosis of reversion to fetal consciousness, e.g., NMS.Usually, the sample and control or predetermined threshold levels arefrom the same tissue types.

Alternatively, if the levels of the one or more measured neurosteroid(e.g., pregnane) compounds in the test biological sample are less thanthe levels of the one or more measured neurosteroid (e.g., pregnane)compounds in the positive control sample or the predetermined thresholdlevel, then a diagnosis of reversion to fetal consciousness, e.g., NMS,is generally not indicated. Likewise, if the levels of the one or moremeasured neurosteroid (e.g., pregnane) compounds in the test biologicalsample are equivalent to or less than a negative or normal controlsample or the predetermined threshold level, then a diagnosis ofreversion to fetal consciousness, e.g., NMS, is not indicated.

In varying embodiments, the predetermined threshold level for theneurosteroids selected from allopregnanolone(3α-hydroxy-5α-pregnan-20-one or 3α,5α-tetrahydroprogesterone; alsoabbreviated as THP or THPROG), allopregnanolone sulfate,5α-dihydroprogesterone (DHP), 17-hydroxypregnenolone,17-hydroxyprogesterone, pregnanediol, pregnenolone sulfate,1,4-androstadiene-3,17-1, boldenone, boldenone sulfate, 5-αdihydroandrolone, 5-α dihydrotestosterone, 5-β dihydrotestosterone,epinandrolone, 17-α estradiol, 17β estradiol, 17-β estradiol sulfate,5-α-estran-3-β-17 diol, estrone, estrone sulfate,6-α-hydroxyandrostenedione, nandrolone, nandrolone glucuronide,nandrolone sulfate, 19-norandrostenedione, 19-nor-androsterone,19-norepiandrosterone, testosterone, testosterone glucuronide andtestosterone sulfate, adrenocorticotropic hormone (ACTH), adenosine,cortisol and oxytocin is above 0.0001 ng/mL or any detectable level. Invarying embodiments, the predetermined threshold level for androsterone,DHEA, epitestosterone, progesterone and pregnenolone at ages 0-few hoursof age, 24 h, and 48 h old are as shown in Table 1. In varyingembodiments, the threshold value for ACTH is 53 ng/mL), the thresholdvalue for cortisol is 16 ng/mL, the threshold value for adenosine is 0.3ng/mL), and the threshold value for oxytocin is 1.1 ng/mL.

TABLE 1 Steroid (ng/mL) 0 h 24 h 48 h androstenedione 0.6 0.3 0.0001(any detectable level) DHEA 50 25 8 epitestosterone 0.2 0.05 0.004progesterone 4 0.0001 (any 0.0001 (any detectable level) detectablelevel) pregnenolone 1200 193 104

Illustrative blood, serum or plasma threshold levels of pregnenolone ina neonate equine at 0 h, 24 h and 48 h post birth are at least about6000 ng/ml at 0-24 h post birth, at least about 550 ng/ml at 24-48 hpost birth and at least about 315 ng/ml at about 48 h post birth andthereafter. In varying embodiments, the elevated pregnenolone levels areabove about 12,900 ng/ml under 24 hours post birth, above about 3470ng/ml at 24-28 hours post birth and/or above about 3420 ng/ml at orafter 48 hours post birth. In varying embodiments, the elevatedprogesterone levels are above about 10.5 ng/ml under 24 hours postbirth, above about 0.8 ng/ml at 24-28 hours post birth and/or aboveabout 0.1 ng/ml at or after 48 hours post birth. In varying embodiments,the elevated progesterone levels are above about 9.7 ng/ml at 24-28hours post birth and/or above about 14.3 ng/ml at or after 48 hours postbirth.

c. Providing a Diagnosis

Upon a positive determination of elevated levels of one or moreneurosteroids (e.g., pregnanes) in the biological sample of the subject,a positive diagnosis of reversion to fetal consciousness (e.g., NMS) maybe provided. The subject may also be exhibiting one or more symptomsconsistent with reversion to fetal consciousness (e.g., NMS). In varyingembodiments, the diagnosis is provided to the parents of a human neonateor to the guardian/owner of a non-human neonate. In addition toproviding a positive diagnosis, options for treatment may be provided,e.g., to prevent, reduce, inhibit, mitigate and/or reverse one or moresymptoms associated with reversion to fetal consciousness (e.g., NMS).

4. Methods of Treatment

Upon a positive determination of elevated levels of one or moreneurosteroids (e.g., pregnanes) and a diagnosis of reversion to fetalconsciousness, the neonate can be treated by pharmacological (e.g.,administration of an effective amount of a 5α reductase inhibitor) orphysical (e.g., squeezing and/or hugging) means, or a combination ofboth pharmacological and physical methods.

a. Pharmacological Methods

In some embodiments, a neonate determined to be suffering from reversionto fetal consciousness, e.g., NMS, is administered an effective amountof a 5α reductase inhibitor. Illustrative 5α reductase inhibitors of useinclude, e.g., alfatradiol, dutasteride, finasteride, bexlosteride,epristeride, izonsteride, lapisteride, turosteride, and analogs, saltsand mixtures thereof.

i. Formulation

The one or more 5α reductase inhibitors, and salts and analogs thereof,can be administered orally, parenterally, (intravenously (IV),intramuscularly (IM), depo-IM, subcutaneously (SQ), and depo-SQ),sublingually, intranasally (inhalation), intrathecally, transdermally(e.g., via transdermal patch), topically, ionophoretically or rectally.In varying embodiments, the dosage form is selected to facilitatedelivery to the brain (e.g., passage through the blood brain barrier).In this context it is noted that the one or more 5α reductase inhibitorsdescribed herein can be readily delivered to the brain. Dosage formsknown to those of skill in the art are suitable for delivery of the oneor more 5α reductase inhibitors.

Compositions are provided that contain therapeutically effective amountsof the compound. The one or more 5α reductase inhibitors are preferablyformulated into suitable pharmaceutical preparations such as tablets,capsules, or elixirs for oral administration or in sterile solutions orsuspensions for parenteral administration. Typically the one or more 5αreductase inhibitors described above are formulated into pharmaceuticalcompositions using techniques and procedures well known in the art.

The one or more 5α reductase inhibitors can be administered in the“native” form or, if desired, in the form of salts, esters, amides,prodrugs, derivatives, and the like, provided the salt, ester, amide,prodrug or derivative is suitable pharmacologically effective, e.g.,effective in the present method(s). Salts, esters, amides, prodrugs andother derivatives of the active agents can be prepared using standardprocedures known to those skilled in the art of synthetic organicchemistry and described, for example, by March (1992) Advanced OrganicChemistry; Reactions, Mechanisms and Structure, 4th Ed. N.Y.Wiley-Interscience.

Methods of formulating such derivatives are known to those of skill inthe art. For example, the disulfide salts of a number of delivery agentsare described in PCT Publication WO 2000/059863 which is incorporatedherein by reference. Similarly, acid salts of therapeutic peptides,peptoids, or other mimetics, and can be prepared from the free baseusing conventional methodology that typically involves reaction with asuitable acid. Generally, the base form of the drug is dissolved in apolar organic solvent such as methanol or ethanol and the acid is addedthereto. The resulting salt either precipitates or can be brought out ofsolution by addition of a less polar solvent. Suitable acids forpreparing acid addition salts include, but are not limited to bothorganic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvicacid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, orotic acid, and the like, aswell as inorganic acids, e.g., hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like. An acidaddition salt can be reconverted to the free base by treatment with asuitable base. Certain particularly preferred acid addition salts of theactive agents herein include halide salts, such as may be prepared usinghydrochloric or hydrobromic acids. Conversely, preparation of basicsalts of the active agents of this invention are prepared in a similarmanner using a pharmaceutically acceptable base such as sodiumhydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide,trimethylamine, or the like. In certain embodiments basic salts includealkali metal salts, e.g., the sodium salt, and copper salts.

For the preparation of salt forms of basic drugs, the pKa of thecounterion is preferably at least about 2 pH lower than the pKa of thedrug. Similarly, for the preparation of salt forms of acidic drugs, thepKa of the counterion is preferably at least about 2 pH higher than thepKa of the drug. This permits the counterion to bring the solution's pHto a level lower than the pHmax to reach the salt plateau, at which thesolubility of salt prevails over the solubility of free acid or base.The generalized rule of difference in pKa units of the ionizable groupin the active pharmaceutical ingredient (API) and in the acid or base ismeant to make the proton transfer energetically favorable. When the pKaof the API and counterion are not significantly different, a solidcomplex may form but may rapidly disproportionate (e.g., break down intothe individual entities of drug and counterion) in an aqueousenvironment.

Preferably, the counterion is a pharmaceutically acceptable counterion.Suitable anionic salt forms include, but are not limited to acetate,benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate,edetate, edisylate, estolate, fumarate, gluceptate, gluconate,hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate,maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate,napsylate, nitrate, pamoate (embonate), phosphate and diphosphate,salicylate and disalicylate, stearate, succinate, sulfate, tartrate,tosylate, triethiodide, valerate, and the like, while suitable cationicsalt forms include, but are not limited to aluminum, benzathine,calcium, ethylene diamine, lysine, magnesium, meglumine, potassium,procaine, sodium, tromethamine, zinc, and the like.

In various embodiments preparation of esters typically involvesfunctionalization of hydroxyl and/or carboxyl groups that are presentwithin the molecular structure of the active agent. In certainembodiments, the esters are typically acyl-substituted derivatives offree alcohol groups, e.g., moieties that are derived from carboxylicacids of the formula RCOOH where R is alky, and preferably is loweralkyl. Esters can be reconverted to the free acids, if desired, by usingconventional hydrogenolysis or hydrolysis procedures.

Amides can also be prepared using techniques known to those skilled inthe art or described in the pertinent literature. For example, amidesmay be prepared from esters, using suitable amine reactants, or they maybe prepared from an anhydride or an acid chloride by reaction withammonia or a lower alkyl amine.

The one or more 5α reductase inhibitors or a physiologically acceptablesalt or ester can be compounded with a physiologically acceptablevehicle, carrier, excipient, binder, preservative, stabilizer, flavor,etc., in a unit dosage form as called for by accepted pharmaceuticalpractice. The amount of active substance in those compositions orpreparations is such that a suitable dosage to prevent, reduce and/ormitigate reversion to fetal consciousness, e.g., NMS, without inducingor eliciting adverse side effects in the neonate. In varyingembodiments, the one or more 5α reductase inhibitors are administered ata dose that is in the range of about 10% to about 50% of a doseadministered to an adult mammal of the same species, e.g., in the rangeof about 25 to about 30% of a dose that induces or elicits desiredpharmacological effects in an adult mammal of the same species, e.g.,about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of a doseadministered to an adult mammal of the same species. The term “unitdosage from” refers to physically discrete units suitable as unitarydosages for mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient.

Determination of an effective amount is well within the capability ofthose skilled in the art. Generally, an efficacious or effective amountof one or more 5α reductase inhibitors is determined by firstadministering a low dose or small amount of a 5α reductase inhibitor andthen incrementally increasing the administered dose or dosages, until adesired effect is observed in the treated subject with minimal or notoxic side effects. Applicable methods for determining an appropriatedose and dosing schedule for administration of a combination of thepresent invention are described, for example, in Brunton, et al.,Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12thEdition, 2010, McGraw-Hill Professional; in a Physicians' Desk Reference(PDR), 66^(th) Edition, 2012; in Loyd, et al., Remington: The Scienceand Practice of Pharmacy, 22^(nd) Ed., 2012, Pharmaceutical Press; andin Martindale: The Complete Drug Reference, 37^(th) Ed., Sweetman, 2011,Pharmaceutical Press., each of which are hereby incorporated herein byreference.

In varying embodiments, the dose of 5α reductase inhibitor administeredto a neonate equine is about 1×, 2×, 5× or 10× the dose of 5α reductaseinhibitor administered to an adult human. For example, in oneembodiment, a dose of 0.1 mg/kg (5 mg total dose) dutasteride isadministered to a neonate equine.

To prepare compositions, the one or more 5α reductase inhibitors aremixed with a suitable pharmaceutically acceptable carrier. Upon mixingor addition of the one or more 5α reductase inhibitors, the resultingmixture may be a solution, suspension, emulsion, or the like. Liposomalsuspensions may also be suitable as pharmaceutically acceptablecarriers. These may be prepared according to methods known to thoseskilled in the art. The form of the resulting mixture depends upon anumber of factors, including the intended mode of administration and thesolubility of the one or more 5α reductase inhibitors in the selectedcarrier or vehicle. The effective concentration is sufficient forlessening or ameliorating at least one symptom of the disease, disorder,or condition treated and may be empirically determined.

Pharmaceutical carriers or vehicles suitable for administration of theone or more 5α reductase inhibitors provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration. In addition, the active materials canalso be mixed with other active materials that do not impair the desiredaction, or with materials that supplement the desired action, or haveanother action. The one or more 5α reductase inhibitors may beformulated as the sole pharmaceutically active ingredient in thecomposition or may be combined with other active ingredients.

Where the one or more 5α reductase inhibitors exhibit insufficientsolubility, methods for solubilizing may be used. Such methods are knownand include, but are not limited to, using cosolvents such asdimethylsulfoxide (DMSO), using surfactants such as Tween™, anddissolution in aqueous sodium bicarbonate. Derivatives of the one ormore 5α reductase inhibitors, such as salts or prodrugs may also be usedin formulating effective pharmaceutical compositions.

The concentration of the one or more 5α reductase inhibitors iseffective for delivery of an amount upon administration that lessens orameliorates at least one symptom of the disorder for which the one ormore 5α reductase inhibitors are administered and/or that is effectivein a prophylactic context. Typically, the compositions are formulatedfor single dosage (e.g., daily) administration.

The one or more 5α reductase inhibitors may be prepared with carriersthat protect them against rapid elimination from the body, such astime-release formulations or coatings. Such carriers include controlledrelease formulations, such as, but not limited to, microencapsulateddelivery systems. The one or more 5α reductase inhibitors are includedin the pharmaceutically acceptable carrier in an amount sufficient toexert a therapeutically useful effect in the absence of undesirable sideeffects on the patient treated. The therapeutically effectiveconcentration may be determined empirically by testing the one or more5α reductase inhibitors in known in vitro and in vivo model systems forthe treated disorder (e.g., reversion to fetal consciousness, e.g.,NMS,). A therapeutically or prophylactically effective dose can bedetermined by first administering a low dose, and then incrementallyincreasing until a dose is reached that achieves the desired effect withminimal or no undesired side effects.

In various embodiments, the one or more 5α reductase inhibitors and/oranalogs thereof can be enclosed in multiple or single dose containers.The one or more 5α reductase inhibitors and compositions can be providedin kits, for example, including component parts that can be assembledfor use. For example, the one or more 5α reductase inhibitors can beprovided in lyophilized form and a suitable diluent may be provided asseparated components for combination prior to use. A kit may include oneor more 5α reductase inhibitors and a second therapeutic agent forco-administration. The one or more 5α reductase inhibitors and secondtherapeutic agent may be provided as separate component parts. A kit mayinclude a plurality of containers, each container holding one or moreunit dose of the compounds. The containers are preferably adapted forthe desired mode of administration, including, but not limited to foodcompositions (e.g., kibble, pellets, cookies), tablets, gel capsules,sustained-release capsules, and the like for oral administration; depotproducts, pre-filled syringes, ampules, vials, and the like forparenteral administration; and patches, medipads, creams, and the likefor topical administration.

The concentration and/or amount of the one or more 5α reductaseinhibitors in the drug composition will depend on absorption,inactivation, and excretion rates of the active compound, the dosageschedule, and amount administered as well as other factors known tothose of skill in the art.

The one or more 5α reductase inhibitors may be administered at once, ormay be divided into a number of smaller doses to be administered atintervals of time. It is understood that the precise dosage and durationof treatment is a function of the disease being treated and may bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed compositions.

If oral administration is desired, the one or more 5α reductaseinhibitors can be provided in a formulation that protects it from theacidic environment of the stomach. For example, the composition can beformulated in an enteric coating that maintains its integrity in thestomach and releases the one or more 5α reductase inhibitors in theintestine. The composition may also be formulated in combination with anantacid or other such ingredient.

Oral compositions will generally include an inert diluent or an ediblecarrier and may be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, the one ormore 5α reductase inhibitors can be incorporated with excipients andused in the form of food compositions (e.g., kibble, pellets, cookies),tablets, capsules, or troches. Pharmaceutically compatible bindingagents and adjuvant materials can be included as part of thecomposition.

In various embodiments, the food compositions (e.g., kibble, pellets,cookies), tablets, pills, capsules, troches, and the like can containany of the following ingredients or compounds of a similar nature: abinder such as, but not limited to, gum tragacanth, acacia, corn starch,or gelatin; an excipient such as microcrystalline cellulose, starch, orlactose; a disintegrating agent such as, but not limited to, alginicacid and corn starch; a lubricant such as, but not limited to, magnesiumstearate; a gildant, such as, but not limited to, colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; and aflavoring agent such as peppermint, methyl salicylate, or fruitflavoring.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials, whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The one or more 5α reductase inhibitorscan also be administered as a component of an elixir, suspension, syrup,wafer, chewing gum or the like. A syrup may contain, in addition to theone or more 5α reductase inhibitors, sucrose as a sweetening agent andcertain preservatives, dyes and colorings, and flavors.

The active materials can also be mixed with other active materials thatdo not impair the desired action, or with materials that supplement thedesired action.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components: asterile diluent such as water for injection, saline solution, fixed oil,a naturally occurring vegetable oil such as sesame oil, coconut oil,peanut oil, cottonseed oil, and the like, or a synthetic fatty vehiclesuch as ethyl oleate, and the like, polyethylene glycol, glycerine,propylene glycol, or other synthetic solvent; antimicrobial agents suchas benzyl alcohol and methyl parabens; antioxidants such as ascorbicacid and sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid (EDTA); buffers such as acetates,citrates, and phosphates; and agents for the adjustment of tonicity suchas sodium chloride and dextrose. Parenteral preparations can be enclosedin ampoules, disposable syringes, or multiple dose vials made of glass,plastic, or other suitable material. Buffers, preservatives,antioxidants, and the like can be incorporated as required.

Where administered intravenously, suitable carriers includephysiological saline, phosphate buffered saline (PBS), and solutionscontaining thickening and solubilizing agents such as glucose,polyethylene glycol, polypropylene glycol, and mixtures thereof.Liposomal suspensions including tissue-targeted liposomes may also besuitable as pharmaceutically acceptable carriers. These may be preparedaccording to methods known for example, as described in U.S. Pat. No.4,522,811.

The one or more 5α reductase inhibitors may be prepared with carriersthat protect the one or more 5α reductase inhibitors against rapidelimination from the body, such as time-release formulations orcoatings. Such carriers include controlled release formulations, suchas, but not limited to, implants and microencapsulated delivery systems,and biodegradable, biocompatible polymers such as collagen, ethylenevinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters,polylactic acid, and the like. Methods for preparation of suchformulations are known to those skilled in the art.

ii. Routes of Administration and Dosing

In various embodiments, the one or more 5α reductase inhibitors and/oranalogs thereof can be administered orally, parenterally (IV, IM,depo-IM, SQ, and depo-SQ), sublingually, intranasally (inhalation),intrathecally, transdermally (e.g., via transdermal patch), topically,or rectally. Dosage forms known to those skilled in the art are suitablefor delivery of the one or more 5α reductase inhibitors and/or analogsthereof.

In various embodiments, the one or more 5α reductase inhibitors and/oranalogs thereof may be administered enterally or parenterally. Whenadministered orally, the compounds can be administered in usual dosageforms for oral administration as is well known to those skilled in theart. These dosage forms include the usual solid unit dosage forms oftablets and capsules as well as liquid dosage forms such as solutions,suspensions, and elixirs. When the solid dosage forms are used, it ispreferred that they be of the sustained release type so that thecompound needs to be administered only once or twice daily.

The oral dosage forms can be administered to the patient 1, 2, 3, or 4times daily. It is preferred that the compound be administered eitherthree or fewer times, more preferably once or twice daily. Hence, it ispreferred that the compound be administered in oral dosage form. It ispreferred that whatever oral dosage form is used, that it be designed soas to protect the compound from the acidic environment of the stomach.Enteric coated tablets are well known to those skilled in the art. Inaddition, capsules filled with small spheres each coated to protect fromthe acidic stomach, are also well known to those skilled in the art.

The one or more 5α reductase inhibitors and/or analogs thereof may alsobe advantageously delivered in a nano crystal dispersion formulation.Preparation of such formulations is described, for example, in U.S. Pat.No. 5,145,684. Nano crystalline dispersions of HIV protease inhibitorsand their method of use are described in U.S. Pat. No. 6,045,829. Thenano crystalline formulations typically afford greater bioavailabilityof drug compounds.

In various embodiments, the one or more 5α reductase inhibitors and/oranalogs thereof can be administered parenterally, for example, by IV,IM, depo-IM, SC, or depo-SC.

In various embodiments, the compounds and/or analogs thereof can beadministered sublingually. When given sublingually, the compounds and/oranalogs thereof can be given one to four times daily in the amountsdescribed above for IM administration.

In various embodiments, the compounds and/or analogs thereof can beadministered intranasally. When given by this route, the appropriatedosage forms are a nasal spray or dry powder, as is known to thoseskilled in the art. The dosage of compound and/or analog thereof forintranasal administration is the amount described above for IMadministration.

In various embodiments, compound and/or analogs thereof can beadministered intrathecally. When given by this route the appropriatedosage form can be a parenteral dosage form as is known to those skilledin the art. The dosage of compound and/or analog thereof for intrathecaladministration is the amount described above for IM administration.

In certain embodiments, the compound and/or analog thereof can beadministered topically. When given by this route, the appropriate dosageform is a cream, ointment, or patch. Because the amount that can bedelivered by a patch is limited, two or more patches may be used. Thenumber and size of the patch is not important, what is important is thata therapeutically effective amount of compound be delivered as is knownto those skilled in the art. The compound can be administered rectallyby suppository as is known to those skilled in the art.

It should be apparent to one skilled in the art that the exact dosageand frequency of administration will depend on the particular conditionbeing treated, the severity of the condition being treated, the age,weight, general physical condition of the particular patient, and othermedication the individual may be taking as is well known toadministering physicians who are skilled in this art.

b. Physical Methods

In some embodiments, a neonate determined to be suffering from reversionto fetal consciousness, e.g., NMS, is subject to squeezing or hugging ofthe mid-thorax region. In varying embodiments, the squeezing and/orhugging applies uniform pressure along the mid-thorax region with apressure and for a time period sufficient to mimic passage through thebirth canal appropriate to the species of the subject. For example, insome embodiments, the applied pressure of the squeezing is maintainedfor at least 10 minutes, e.g., at least 15, 20, 25, 30, 35, 40, 45, 50,55 or 60 minutes, as appropriate or necessary. The pressure may beapplied one or more intervals, e.g., 1, 2, 3, 4, 5, or more intervals,as appropriate or necessary.

When the subject is a human, skin-on-skin contact is preferred, with theneonate's head placed near the heart of the person applying squeezing orhugging pressure (e.g., the mother). So-called Kangaroo Mother Care isknown in the art and reviewed, e.g., in Moore, et al., Cochrane DatabaseSyst Rev. (2012) May 16; (5):CD003519; Bailey, Br J Hosp Med (Lond).(2012) 73(5):278-81; Bulfone, et al., Prof Inferm. (2011) 64(2):75-82;Conde-Agudelo, et al., Cochrane Database Syst Rev. (2011) March 16; (3):CD002771; and Lawn, et al., Int J Epidemiol. (2010) 39 Suppl 1:i144-54.

In varying embodiments, pressure is applied by a rope or a cuff or asleeve. For example, substantially uniform pressure can be applied byencircling the mid-thorax region of the neonate in an inflatable cuff orsleeve. In varying embodiments, the mid thorax region of the neonate isencircled and subject to pressure with an inflatable cuff or sleevesystem. The inflatable cuff or sleeve can comprise a pneumatic inflatingsleeve, e.g., similar to a blood pressure cuff. The cuff or sleeve canbe constructed of a material or coated with a material that does notabrade or injure the neonate upon contact. The cuff or sleeve can beinflated to a predetermined pressure (e.g., to about 10 psi or less)over the proximal and mid thorax of the neonate (e.g., foal) andmaintained at a pressure and for time period sufficient to mimic thebirth canal, e.g., sufficient to provide deep touch pressure. In varyingembodiments, the inflated cuff or sleeve provides substantially uniformpressure to the mid-thorax of the neonate. As appropriate, theinflatable cuff or sleeve can be inflated using a foot pump or anautomated pump motor. The inflatable cuff or sleeve can be kept in placeby use of straps. In varying embodiments, for inflatable cuffs orsleeves designed for use on quadruped mammals (e.g., equine neonates),the device can have straps designed to around the neonate's neck andbetween the front legs. The inflatable cuff or sleeve is provided in asize appropriate to the size of the species being treated, such thatwhen inflated, the cuff or sleeve provides substantially uniformpressure to the mid-thorax region of the neonate. The inflatable cuff orsleeve can be designed to be a reusable device.

In varying embodiments, multiple cuff or sleeve devices are used intandem. For example, two pneumatic sleeves can be placed over the midbody, a first over the heart—girth area and a second over the caudalarea—anywhere from ribs to abdomen. Each sleeve has a pneumaticinflation device to ensure constant pressure that does induce somnolencebut does not impair ventilation (e.g., to about 10 psi or less). For useon equines and other quadrupeds, the sleeve can be placed on the neonate(e.g., the foal) when standing (FIG. 3). For applying the first device,a strap (e.g., canvas, leather or any appropriate material) can beplaced around the neonate's neck to prevent the device from slidingcaudally. In varying embodiments, the device can be secured around thethoracic circumference of the neonate through any appropriate means(e.g., by looping a strap, a metal hinge, a hook, a clasp, a belt). Inone embodiment, the strap is folded back upon itself and can be pulledsnugly or tightly and attached to Velcro. A hand or foot operated orautomated airpump can be used to inflate the pneumatic sleeve,(inflatable sleeve in FIG. 4). The sleeve can be inflated to an internalpressure sufficient to exert a substantial squeezing pressure on theneonate. In varying embodiments, the cuffs or sleeves are inflated toabout 10 psi. The pressure can be checked with an air gauge. The seconddevice can be similarly applied, as illustrated in FIG. 3. The neonatethen becomes laterally recumbent and stays that way for an appropriateperiod of time (e.g., 20-30 minutes). FIG. 5 shows the device attachedon an equine foal with the strap around the neck to hold it in place.Upon completion of the squeezing procedure, the air is released from thesleeve and the sleeve is detached and the foal is allowed to wake up.Heart-girth measurements vary depending on species and breed but thedevice can be readily designed to fit any neonate, e.g., any equine foalof varying breeds less than 1 week of age. Typically, the cuffs orsleeves range from about 26 to about 48 inches in length, e.g.,sufficient to encircle the circumference of a neonatal equine over theheart-girth area.

5. Methods of Monitoring

The elevated levels of one or more neurosteroids (e.g., pregnanes, e.g.,pregnenolone) correlated with a syndrome of reversion to fetalconsciousness (e.g., NMS) allows for the course of treatment formitigating and/or reversing reversion to fetal consciousness (e.g., NMS)to be monitored. For methods of monitoring, one or more test biologicalsamples are obtained from a subject undergoing treatment for mitigatingand/or reversing reversion to fetal consciousness (e.g., NMS). Asappropriate, biological samples are obtained from the subject at varioustime points, e.g., before, during, and/or after a course of treatment(e.g., physical and/or pharmacological). Levels of one or moreneurosteroid (e.g., pregnane) compounds in the biological sample (e.g.,pregnenolone) are then determined and compared to a reference biologicalsample which includes known levels of the one or more neurosteroids(e.g., pregnanes). In varying embodiments, the reference biologicalsample is from an untreated subject either known to suffer fromreversion to fetal consciousness (e.g., NMS) or known to be normal.Generally, test and control biological samples are the same (e.g.,blood, serum, plasma, urine).

In embodiments where the reference biological sample is from a normalcontrol subject, a similarity in the levels of the one or more measuredneurosteroids (e.g., pregnanes) in the test biological sample and thereference biological sample indicates that the treatment is efficacious.However, increased or elevated levels of one or more neurosteroids(e.g., pregnanes) in the test biological sample taken at one or moretime points in comparison to levels of the same one or moreneurosteroids (e.g., pregnanes) in a normal control reference biologicalsample indicates a less favorable clinical outcome or prognosis.Similarly, if the reference biological sample is from a control subjectknown to suffer from reversion to fetal consciousness (e.g., NMS), adecrease or reduction in levels of one or more of the measuredneurosteroids (e.g., pregnanes, e.g., pregnenolone) in the testbiological sample in comparison to levels of the same one or morepregnanes in the positive control reference biological sample indicatesthat the treatment is efficacious, while similar or elevated levels ofthe one or more neurosteroids (e.g., pregnanes) in the test populationand the positive control reference biological sample indicates a lessfavorable clinical outcome or prognosis.

Additionally, the levels of the one or more neurosteroids (e.g.,pregnanes) determined in a biological sample from a subject obtainedafter treatment (i.e., post-treatment levels) can be compared to thelevels of the one or more neurosteroids (e.g., pregnanes) determined ina biological sample from the same subject obtained prior to treatmentonset (i.e., pre-treatment levels). A decrease in the levels of the oneor more neurosteroids (e.g., pregnanes) in a post-treatment sampleindicates that the treatment is efficacious while an increase ormaintenance in the levels of the one or more neurosteroids (e.g.,pregnanes) in the post-treatment sample indicates a less favorableclinical outcome or prognosis.

As used herein, the term “efficacious” indicates that the treatmentleads to a reduction in the levels of the one or more measuredneurosteroids (e.g., pregnanes) in a subject that are associated with,correlative and/or indicative of reversion to fetal consciousness (e.g.,NMS). When a treatment of interest is applied prophylactically, the term“efficacious” means that the treatment retards or prevents symptoms ofreversion to fetal consciousness (e.g., NMS) from forming or retards,prevents, or alleviates one or more symptoms of reversion to fetalconsciousness.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Allopregnanolone Infusion Induced Neurobehavioural Alterationsin a Neonatal Foal Materials and Methods

A healthy neonatal 50 kg Quarter Horse colt from the research herd atthe School of Veterinary Medicine, University of California, Davis wasselected for the infusion. The foal was born from a healthy QuarterHorse mare with a normal gestational length without complications, andnormal observed parturition except that the placental passage wasprolonged. Although retention of the placenta for 7 h post birth is notconsidered normal by most standards, the placenta was evaluated anddetermined to be complete and normal. Further, the mare did not displayany signs or complications associated with placental retention. The foalwas deemed healthy based upon complete physical and neurologicalexaminations immediately post birth. The foal exhibited normal adaptivebehaviour with righting reflex, and time to stand and suckle withinnormal limits. Repeat physical and neurological examinations at age 6 h,immediately prior to infusion, were also normal.

Intravenous catheters were placed aseptically in the right jugular veinfor sample collection and in the left jugular vein for infusion ofallopregnanolone. Allopregnanolone (5 alpha-pregnan-3 alpha-ol-20-one;Steraloids Inc., Newport, R.I., USA) was dissolved in an ethanol-basedsolution to a total concentration of 9 mg/ml. Infused dose andconcentration of allopregnanolone in this foal were determined based onconcentrations reported in in vivo studies in the modulation of the HPAaxis in male rats (Naert, et al., (2007) Psychoneuroendocrinology 32,1062-1078). An initial bolus of 0.05 mg/kg bwt i.v. of allopregnanolonewas given followed by a constant rate infusion (CRI) of 0.02 mg/kgbwt/min using an infusion pump. Based on clinical effects of the initialdosage, a second bolus of 0.1 mg/kg bwt i.v. was given after 5 min andfollowed by a CRI of 0.04 mg/kg bwt/min for 5 min. The infusion wasdiscontinued for 30 min to allow observation of any neurobehavioural(NB) alterations, and then a final bolus of 0.2 mg/kg bwt i.v. wasgiven.

Neurobehavioural alterations were recorded and graded through a NBscoring system developed by the authors for the assessment of foals withNMS (Table 2). From preliminary work, foals with NMS had scores >8 froma range of 0 (normal foal) to 20 (comatose with paroxysmal activity).Mentation was defined as: normal if the foal was alert and responsive;quiet to obtunded if the foal was apparently lethargic but responsive toexternal stimuli (e.g., touch, sound); stuporous if level ofconsciousness was decreased but responsive to painful stimuli (e.g.pinching skin with haemostats); and comatose if the foal lostconsciousness and was unresponsive to any stimuli. Paroxysmal activitywas defined as abnormal events such as seizures or seizure-likeactivity, rhythmic limb movements, tremors or paddling. The NB scoreswere calculated at 5 min intervals throughout the infusion period.Following the infusion, the foal was observed hourly for the first 6 hand then at 12 h intervals for 2 days.

TABLE 2 Neurobehavioral scoring system. Parameter 0 1 2 3 Mentation andNormal, bright, Mildly obtunded, Moderately obtunded, Severely obtundedwith reaction to alert responsive slightly decreased or moderatelydecreased hyper-reactivity to stimuli increased reactivity or increasedreactivity stimuli, to comatose to stimuli to stimuli Ability to standStands unassisted Stands with minimal Stands with marked Unable to standassistance support Bonding to mare Actively bonds Slightly reducedAimless wandering or Unaware of mare with and follows interaction withmare periods of reduced mare responsiveness to Ability to nurse Latcheson and Searches out teat but Weak, ineffective No suckling nurseseffectively does not nurse suckling vigorously Paroxysms None 0 None,limb stretching, Seizures or seizure-like paddling activity Ear positionErect 0 Ears partially erect Floppy, no tone Paroxysms is defined asepisodes of abnormal activity.

Heparinised blood samples were collected at birth, age 6 h and at 15 minintervals during the infusion. Blood was immediately centrifugedfollowing collection and plasma stored at −80° C. until analysed byliquid chromatography mass spectrometry (LC-MS) utilising on-line sampleextraction by turbulent flow chromatography (TFC) and detection byselect reaction monitoring (SRM) on a triple quadrupole massspectrometer. Samples were diluted 2:1 with water fortified with 4internal standards: D3-testosterone, D3-boldenone, D7-androstenedioneand D3-testosterone sulphate. Analytes were separated by liquidchromatography using a Thermo TLX-2 TFC system with a Thermo Cyclone Pextraction column and an ACE C18 analytical column. Analytes wereintroduced by electrospray ionisation to a Thermo TSQ Vantagetriple-quadrupole mass spectrometer operating in both negative andpositive modes. Free concentrations of 34 steroids were monitored in oneanalytical method over a 24-min run time. Detection and quantitation wasaccomplished using 3 or more SRM transitions per compound for allcompounds other than 17-hydroxy pregnenolone where single ion monitoring(SIM) was utilised. This method was validated and the following assessedfor each analyte: linearity, limit of detection, limit of quantitation,accuracy, precision, matrix effects, extraction recovery and potentialendogenous interferences. The following steroids were analysed:allopregnanolone, dehydroepiandrosterone (DHEA), 5-alphadihydroprogesterone, 17-hydroxy pregnenolone, pregnenolone, pregnanedioland progesterone. A diagram of the relation of these neurosteroids isshown in FIG. 1.

For NB comparison, a second age-matched clinically healthy neonatalQuarter Horse colt was infused with 99.9% ethanol diluted with 0.9%saline to a final concentration of 5% ethanol without allopregnanolone.Infusion of this solution followed the same protocol (dosage [based on5% ethanol] and rate) of administration as for the first foal. The studywas approved by an Animal Care and Use protocol from University ofCalifornia, Davis.

Results

Prior to the allopregnanolone infusion, the colt was bright, alert andresponsive (NB score of 0; File 51 (published on online with Madigan, etal., Equine Veterinary Journal 44, Suppl. 41 (2012) 109-112; accessibleon the internet athttp://onlinelibrary.wiley.com/doi/10.1111/j.2042-3306.2011.00504.x/full).Infusion of 0.05 mg/kg bwt allopregnanolone followed by a CRI at 0.02mg/kg bwt/min resulted in signs of sedation and decreased responsivenessto the environment (NB score of 14). Infusion of higher concentrationsof allopregnanolone (0.1 and 0.2 mg/kg bwt) resulted in dramatic NBeffects with the foal becoming recumbent, stuporous, unresponsive to themare, environment, sound and tactile stimulation (NB score of 16; FileS2-S4 (published on online with Madigan, et al., Equine VeterinaryJournal 44, Suppl. 41 (2012) 109-112; accessible on the internet athttp://onlinelibrary.wiley.com/doi/10.1111/j0.2042-3306.2011.00504.x/full)). Clinical signs persisted during theconstant rate infusion. Within 8 min of cessation of the infusion thefoal began to show signs of increased responsiveness. By 15 min aftercessation of the infusion or the final bolus the foal was standing butcontinued to show clinical signs of mild obtundation, reducedcoordination and poor udder seeking ability (NB score of 9; File S5(published on online with Madigan, et al., Equine Veterinary Journal 44,Suppl. 41 (2012) 109-112; accessible on the internet athttp://onlinelibrary.wiley.com/doi/10.1111/j0.2042-3306.2011.00504.x/full)). The foal appeared normal by 30 minafter infusion of the neurosteroid (NB score of 0; File S6 (published ononline with Madigan, et al., Equine Veterinary Journal 44, Suppl. 41(2012) 109-112; accessible on the internet athttp://onlinelibrary.wiley.com/doi/10.1111/j0.2042-3306.2011.00504.x/full)). No long-term NB effects were observedfollowing the infusion. The control foal's NB scores were unchangedthroughout the infusion. Steroid concentrations from Foal 1 are detailedin Table 3; these were not measured in the control foal due to lack ofNB alterations, cost and probability of undetectable concentrations ofneurosteroids. Due to an increase in dehydroepiandrosterone (DHEA)concentrations between birth and age 6 h, luteinising hormone (LH)concentrations at various time points were measured to investigate ifthis rise was due to production by the testis. However, there was nochange in LH concentration.

TABLE 3 Steroid concentrations and NB score (0-20) in a healthy foalinfused with allopregnanolone Age Neurobehavior ACTH Cortisol 17-OH(hours) Time point score ( pmol/L) (nmol/L) DHEA pregnenolone 0 10minutes post-birth 0 NP NP 37,940 1,295,070 6 Pre-infusion 0 3.4 6486,593 91,877 6 ¼ Bolus 0.05 mg/kg IV, 14 2.1 58 74,475 73,982 CRI 0.02mg/kg/min 6 ½ 15 minutes post initial 8 2.6 55 1,232,216 95,275 infusion6 ¾ Bolus 0.2 mg/kg IV 15 4.7 40 56,910 57,581 Age 5-α (hours)Progesterone Pregnenolone Dihydroprogesterone AllopregnanolonePregnanediol 0 3,126 3,074,455 ND ND 110,674 6 880 1,279,409 ND ND67,134 6 ¼ 776 1,252,565 18,065 478,909 63,241 6 ½ 1,189 1,206,68418,842 126,566 73,497 6 ¾ 646 1,144,544 28,406 466,208 79,574 Allconcentrations are in pg/mL unless stated, neurobehavioral score (0-18),DHEA = dehydroepiandrosterone, NP = not performed, ND = not detected.

Discussion

Infusion of allopregnanolone to a healthy foal in this study producedmarked NB effects. This is consistent with the clinical use of certainsteroidal drugs, such as alphaxalone, as anaesthetic agents in male rats(Naert, et al., (2007) Psychoneuroendocrinology 32:1062-1078).Allopregnanolone in other species has been shown to cross theblood-brain barrier and is thought to mediate its effects in the centralnervous system (CNS) via the GABAA receptor (Zhu, et al., (2001) Br JAnaesth 86:403-412). Infusion of allopregnanolone in this healthy foalprovided evidence that 5-alpha reduced pregnanes can cross theblood-brain barrier and have effects in the CNS. Allopregnanoloneconcentrations peaked in conjunction with maximum NB effects followinginfusion. The rapid recovery from NB alterations with no apparentresidual deficits once the infusion was discontinued, suggested thatallopregnanolone was quickly metabolised in this healthy foal. Similarrapid dampening effects in the CNS and recovery were observed with theuse of the neurosteroid anaesthetic alphaxalone in ponies undergoingcastration (Leece, et al., (2009) Vet Anaesth Analg 36, 480-484). Asallopregnanolone is apparently metabolised rapidly, the clinical signsassociated with NMS in foals would be expected to dissipate rapidly.However, clinical manifestations of NMS can last several days,suggesting ongoing persistent production and release of allopregnanoloneor other neurosteroids responsible for such observations. It is alsounclear what triggers and stops these events in affected foals.Progestagen levels in this healthy foal decreased with age and are inagreement with the results of previous work (Holtan, et al., (1991)JReprod Fert Suppl 44:517-528). The rise in DHEA between birth and age 6h in this foal appeared to be neither testicular nor adrenal in originas determined by constant levels of luteinising hormone and pregnanes,respectively, and was therefore deemed unlikely to be of biologicalrelevance.

Recently, higher plasma concentrations of progesterone, epitestosteroneand androstenedione were found in NMS foals compared with foals withother disorders. Findings from that work (Aleman, et al. (2013) EquineVet J 45:661-665), along with the NB alterations induced by the infusionof allopregnanolone are consistent with the conclusion that NMS is inpart a manifestation of persistent fetal HPA status mediated andsustained by elevated concentrations of progestagens as occurs naturallyin the fetus (Warnes, et al., (2004) Biol Reprod 71:620-628). The fetusmust rapidly change from the quiet suppressed state in utero to one ofarousal, and attempts to rise shortly after birth. A failure of thetransition from the fetal HPA status to immediately post birth signalsto engage the newborn into normal post foaling neurobehaviour may be thecause or involved in part in the pathogenesis of NMS. Further, themeasured neurosteroids and altered neurological status in this studysuggest that neurosteroids readily cross the blood-brain barrier andexert altering CNS effects compatible with NMS in affected foals.Certainly some foals suffer severe birth hypoxia and recover, and havebeen included in the broad description of NMS. However, the recoveryfrom severe birth hypoxia would be expected to be slow and likely tohave residual neurological deficits as documented in all other mammalianspecies suffering severe birth hypoxia (McAuliffe, et al., Brain Res(2006) 1118:208-221). Ongoing production of pregnanes by the foal'sbrain and adrenal glands causes the clinical signs observed in foalswith NMS and rapid recovery of signs with no apparent residual deficitsis compatible with the decline of pregnane-mediated sedative typeeffects (Zhu, et al., (2001) Br J Anaesth 86:403-412).

It is unclear how foals that are normal at birth develop NMS within thefirst 48 h of life. However, a similar mechanism reported in neonatalsheep may occur whereby neonatal stress can increase allopregnanoloneproduction by the brain and release of deoxycorticosterone from theadrenal glands, which the brain metabolises into5α-tetrahydrodeoxycorticosterone (TH-DOC), another CNS depressant(Hirst, et al., (2008) Neurochem Int 52, 602-610). Obtundation, seizuresand hyperaesthesia are common signs of NMS. Whilst the infusedneuroactive steroid allopregnanolone has a dampening effect in the CNS,others within the large spectrum of neurosteroids, including metabolitesof allopregnanolone, have excitatory effects that may be associated withseizures and hyperaesthesia (Rogawski and Reddy, (2004) Neurosteroids:Endogenous modulators of seizure susceptibility. In: Epilepsy:Scientific Foundations of Clinical Practice, Eds: Rho, et al., MarcelDekker, New York. pp 319-355). Neurosteroid concentrations in clinicalNMS are likely to be a far more complex condition than that representedby infusion of one compound.

Example 2 Abnormal Plasma Neurosteroid Concentrations in Ill, NeonatalFoals Presented to the Neonatal Intensive Care Unit Materials andMethods

Foals.

The NMS foal group (n=32; 15 colts and 17 fillies) and the otherneonatal disease foal group (n=12; 4 colts and 8 fillies) were comprisedof foals admitted to the University of California, Davis VeterinaryMedical Teaching Hospital in 2008 and Rossdale and Partners, Newmarket,UK in 2010 and 2011 (Aleman, et al. (2013) Equine Vet J 45:661-665). Tobe included as a foal with NMS, other disorders with a similar clinicalpresentation, such as prematurity and sepsis, were ruled out based on aminimum data base (published sepsis score, complete blood count,chemistry panel, blood gases, indirect blood pressure, central venouspressure, blood culture, urinalysis, abdominal ultrasound, and carpi,tarsi, thoracic and abdominal radiography) (Brewer and Koterba (1988)Equine Vet J 20, 18-22). Foals with a sepsis score of 11 or greater wereadditionally classed as septic (Brewer and Koterba (1988), supra).Historical knowledge of pre-, intra- or post-natal hypoxia was recorded.Clinical signs of NMS included altered mentation (obtunded, stuporous,comatose), decreased bonding to the mare, vocalization, aimlesswandering, hyper- or lack of reactivity to stimuli, seizures, andabnormal ear position. Foals were subjectively scored by the attendingclinician as mild-moderate if able to nurse and ambulate with help, orsevere if recumbent and unable to nurse, even with help. Case details ofNMS foals are given in Table 4. Foals in the other neonatal diseasegroup (sick, non-NMS controls) were randomly selected based on clientconsent and availability of the authors for sample collection. Thesefoals had a variety of clinical diagnoses (Table 5).

TABLE 4 Case histories of neonatal maladjustment syndrome (NMS) foalgroup (n = 32). Age Sepsis Creatinine PMN Case Gender (h) OutcomeClinical history of hypoxia Other diagnosis score Severity (umol/L)(×10⁹/l) 1 M 0 S No No 10 Sev 1800 10.25 2 M 24 S No No 6 M-M 1030 5.443 M 24 S No No 6 M-M 110 7.68 4 M 0 S Caesarean Sepsis 12 Sev 247 2.15 5F 0 S Caesarean No 8 M-M 330 6.25 6 F 0 S Dystocia No 6 M-M 242 10.57 7F 0 S No No 2 M-M 107 12.26 8 F 0 S Dystocia Carpal contracture 7 M-M237 7.55 9 F 0 S No Sepsis, meconium aspiration, rib 12 M-M 576 2.87fractures 10 M 0 E Dystocia Sepsis, anuric renal failure 11 Sev 18128.21 11 F 48 S Bilateral laryngeal paralysis Sepsis, congenitalbilateral 12 M-M 156 5.17 laryngeal paralysis 12 M 6 E Dystocia Sepsis11 Sev 230 5.96 13 F 12 S Dystocia, apnea, blue at birth Sepsis 14 M-M150 9.64 14 F 12 E Premature placental Sepsis 15 Sev 168 1.86 separation15 F 24 S Unobserved foaling No 9 M-M 88 5.31 16 F 4 S Dystocia No 10M-M 168 9.95 17 F 24 S No No 9 Sev 168 11.87 18 M 24 S Unobservedfoaling No 15 Sev 186 2.74 19 M 16 S Dystocia No 9 M-M 274 7.18 20 F 0 SDystocia (GA delivery) Sepsis 11 M-M 124 4.30 21 M 24 S No Meconiumimpaction 3 M-M 83 9.73 22 F 0 E Dystocia No 10 Sev 291 4.41 (economic)23 F 0 S No Dysmaturity, high risk pregnancy 6 M-M 124 4.30 24 F 0 EHistology, placentitis Contracture n/a M-M n/a n/a (due to limbs) 25 F 0S No No 10 Sev 410 5.33 26 M 0 E No Sepsis, gastrocnemius rupture, 12Sev 197 8.16 fetlock varus 27 F 0 S No Sepsis 12 Sev 765 8.93 28 M 0 SDystocia No 7 M-M 281 9.02 29 M 0 S Placentitis, rib fracturesPlacentitis, rib fractures 9 M-M 930 4.44 30 M 0 S Dystocia Sick mare 6M-M 354 7.80 31 M 24 S Rib fracture Sepsis, acute renal failure, rib 14Sev 1528 2.98 fracture 32 M 0 E No Severe meconium impaction 2 Sev 1836.18 (economic) Age = age at presentation; M = male; F = female; E =euthanasia; S = survival; Sev = severe NMS; M-M = mild-moderate NMS; PMN= polymorphonuclear cell count.

TABLE 5 Case histories of sick control foal group (n = 12). Case GenderAge (h) Outcome Clinical Diagnosis Sepsis Score Creatinine (umol/l) PMN(×10⁹/l) 1 F 0 E Prematurity, sepsis, intra-uterine growth restriction12 461 22.61 (economic) 2 F 24 E Dysmature, sepsis, ARDS 16 406 0.95 3 F0 E Severe forelimb contracture n/a n/a n/a 4 F 0 S Dysmature, meconiumimpaction 10 83 4.35 5 F 0 S Dystocia, assisted delivery, rib fractures4 243 6.64 6 F 0 S Dystocia, assisted delivery, angular limb deformities7 158 8.33 7 M 0 S Dysmature, meconium aspiration 14 909 6.5 8 M 24 ERib fractures, traumatic diaphragmatic hernia and 15 159 0.2 colonrupture 9 F 0 S Dysmature, sepsis 12 181 9.48 10 M 0 S Bilateralforelimb contracture 5 127 8.47 11 F 24 S Meconium impaction, urachalrent 5 87 2.69 12 M 0 S Slightly weak 1 118 6.37 Age = age atpresentation; M = male; F = female; E = euthanasia; S = survival; ARDS =acute respiratory distress syndrome; PMN = polymorphonuclear cell count;n/a = not available.

A third group of healthy control neonatal foals (n=10; 4 colts and 6fillies) was recruited from the 2009 and 2010 foal crops at the Centerfor Equine Health, University of California, Davis.

Inclusion criteria for control foals included a term birth (>320 daysgestation) with normal, uncomplicated delivery and physical examination.

All foals were less than 48 hours of age at enrollment into the study.No attempts were made to standardize treatments given to the foalsduring hospitalization. Outcome was recorded as survival to discharge.The study was approved by the University of California InstitutionalAnimal Care and Use and Committee, and client consent was obtained priorto enrollment in the study.

Sample Collection and Analysis.

Heparinized blood was collected from healthy control foals at 0, 24 and48 h following birth. Samples were collected from NMS foals and otherneonatal disease foals after initial stabilization and thereafter at thedesignated 24 and 48 h timepoints as appropriate. For foals presentingat birth, samples were collected within 2 h of parturition.

Whole blood was immediately centrifuged after collection and the plasmastored at −80° C. until analyzed. Plasma was analyzed by liquidchromatography mass spectrometry (LC-MS) utilizing on-line sampleextraction by turbulent flow chromatography (TFC) and detection byselect reaction monitoring (SRM) on a triple quadrupole massspectrometer. Samples were diluted 2:1 with water fortified with fourinternal standards: D3-Testosterone, D3-Boldenone, D7-Androstenedione,and D3-Testosterone Sulphate. Analytes were separated by liquidchromatography using a Thermo TLX-2 TFC system with a Thermo Cyclone Pextraction column and an ACE C18 analytical column. Analytes wereintroduced by electrospray ionization to a Thermo TSQ Vantagetriple-quadrupole mass spectrometer operating in both negative andpositive modes. Free steroid concentrations of 34 steroids weremonitored in one analytical method over a 24 minute run time. Detectionand quantitation was accomplished using 3 or more SRM transitions percompound for all compounds other than 17-hydroxy pregnenolone whereSingle Ion Monitoring (SIM) was utilized. This method was validated andthe following assessed for each analyte: linearity, limit of detection,limit of quantitation, accuracy, precision, matrix effects, extractionrecovery, and potential endogenous interferences. The following steroidswere evaluated: pregnanes including progesterone,17hydroxy-progesterone, 5α-dihydroprogesterone, pregenolone,allopregnanolone, and pregnanediol; androgens and estrogens includingnandrolone sulphate, boldenone sulfate, 17-β estradiol sulphate,testosterone sulphate, 1,4-androstadien-3,17-one, testosteroneglucoronide, 19-norandrostenedione, boldenone, androstenedione,nandrolone, estrone, testosterone, epinandrolone, epitestosterone,6-α-hydroxyandrostenedione, nandrolone glucuronide, 17-β estradiol, 17-αestradiol, 19-norepiandrosterone, dehydroepiandrosterone (DHEA),DHEA-sulphate, 17-hydroxypregnenolone, 5-α dihydronandrolone,5-α-estran-3-β-17-α diol, 5-α dihydrotestosterone, 19-nor-androsterone,5-β dihydrotestosterone, estrone sulphate. These steroids were chosendue to convenience of a pre-existing, extensive steroid panel.

Data Analysis.

Descriptive data are reported as median and ranges. Friedman tests wereused for repeated measures analysis of steroid concentrations of healthyfoals. Kruskal-Wallis tests were used for multiple group comparisons andMann-Whitney tests for non-paired two group comparisons. Non-parametrictests were chosen based on the failure of the data to conform to normaldistributions using a Kolmogorov and Smirnov test. Level of significancewas set at P<0.05.

Results

On presentation, 19 NMS foals were graded as mild-moderate and 13 assevere. Altered states of consciousness of foals with NMS ranged frommildly obtunded to stuporous to comatose.

Several progestagens were detected in extremely low concentrations. Fivesteroids (both pregnanes and androgens) were consistently identifiedamong foal samples; progesterone, pregnenolone, androstenedione, DHEAand epitestosterone. Healthy foals showed progressive, significantdecreases in these steroids over the first 48 h of life (progesteroneP<0.0001; pregnenolone P<0.0001; androstenedione P=0.009; DHEA P=0.006;epitestosterone P=0.004) (FIG. 2). There was no significant differencein healthy foal pregnane or androgen profiles between genders.

Compared to healthy foals, NMS foals showed elevated concentrations ofandrostenedione (P=0.02) and progesterone (P=0.04) at 0 h (within 2 h ofbirth), androstenedione (P=0.0002), DHEA (P=0.001), epitestosterone(P=0.0004), progesterone (P=0.0001) and pregnenolone (P=0.0007) at 24 hof age, and androstenedione (P=0.0008), DHEA (P=0.007), progesterone(P=0.0001) and pregnenolone (P=0.003) at 48 h of age (FIG. 2, Table 6).Sick control foals also had significantly elevated concentrations ofepitestosterone (p=0.03), progesterone (P=0.001) and pregnenolone(P=0.05) at 0 h, androstenedione (P=0.005), DHEA (P=0.003), progesterone(P=0.01) and pregnenolone (P=0.0009) at 24 h and androstenedione(P=0.004), progesterone (P=0.0004) and pregnenolone (P=0.0006) at 48 hcompared to healthy foals (FIG. 2, Table 6). Compared to sick controlfoals, NMS foals had significantly higher concentrations ofepitestosterone at 0 and 24 h of age (P=0.02, 0.002 respectively). Incontrast, sick control foals had significantly higher progesteroneconcentrations than NMS foals at 0 h (P=0.01). Whilst pregnaneconcentrations of sick control foals remained elevated above those ofhealthy foals, their progesterone and pregnenolone concentrationsdecreased significantly (P=0.02, P=0.04 respectively) over the 48 h. Incontrast, steroid concentrations of NMS foals remained elevated andshowed a trend of increasing concentration over time (FIG. 2).

TABLE 6 Median (range) serum steroid concentrations (ng/ml) in healthy,sick control and neonatal maladjustment syndrome (NMS) foals at 48 h ofage. Healthy P value Control Sick Control NMS (Kruskal Steroid (ng/ml)(n = 10) (n = 12) (n = 32) Wallis) Andro- nd^(a) 1.15^(b) 6.56^(b)<0.0001 stenedione (nd-0.51) (nd-7.65) (nd-57.34) DHEA 7.68^(a)101.14^(a, b) 92.98^(b) 0.028 (nd-117.90) (nd-1412.60) (nd-1,511.06)Epites- nd nd 0.53 0.1 tosterone (nd-0.15) (nd-13.20) (nd-11.53) Proges-nd^(a) 6.09^(b) 14.22^(b) <0.0001 terone (nd-0.09) (1.97-14.28)(0.75-73.61) Preg- 103.76^(a) 1119.40^(b) 1922.08^(b) 0.001 nenolone(5.6-313.20) (248.26- (nd-15,917.33) 3,416.08) DHEA =dehydroepiandrosterone; nd = not detectable. Groups with differingsuperscripts are significantly different (Mann-Whitney).

There was no significant difference in pregnane concentrations betweenmild-moderate and severely affected NMS foals. When considering all illfoals admitted to NICU, there was no significant difference in pregnaneconcentrations between survivors and non-survivors, septic andnon-septic individuals and foals with and without a known history ofhypoxia.

Discussion

The results of this study confirm that there are differences in thepregnane profiles of neonatal healthy foals, foals with NMS and foalswith other clinical diagnoses. Pregnane concentrations of healthyneonatal foals declined rapidly, to essentially zero, within 48 h ofbirth in agreement with the study by Houghton et al. (1991). The foetalfoal is subjected to high levels of progesterone and other progestagensin utero (Holtan, et al., (1991) J Pediatr Psychol 44, 517-528), whichis deemed important in providing tonic inhibition of foetal CNS activityand damping movement to prevent maternal damage (Mellor, et al., (2005)Brain Res Rev 49, 455-471). Injections of progesterone or itsmetabolites into the ovine foetal circulation in late gestation reducefoetal electroencephalograph, electrocorticograph and electrooculographactivity, breathing movements and behavioural arousal, whilst inhibitionof placental progesterone enhance these parameters (Crenshaw, et al.,(1966) Nature 212, 842; Crossley, et al., (1997) J. Reprod. Fertil. Dev.9, 767-773; Nicol, et al., (1997)J Endocrinol 152, 379-386; Nicol, etal., (2001) Neurosci Letters 306, 13-16). The loss of placentallyderived precursors at birth and the switch to adrenal or other derivedprecursors causes this dramatic decline in pregnane concentrationsshortly after birth in healthy neonates (Hirst, et al., (2006)Neuroendocrinol 84, 264-274).

Apart from epitestosterone concentrations of sick control foals, foalspresenting ill to the Neonatal Intensive Care Unit (i.e. NMS and sickcontrol foals) had higher concentrations of all measurable pregnanesthan healthy controls within 2 h of birth. Pregnane concentrations ofNMS foals remained elevated over the 48 h time period in contrast tothose of sick control foals which had significantly lower progesteroneand pregnenolone concentrations at 48 h compared to birth. Serial bloodsampling with continued elevation or increasing pregnane concentrationsover 48 h of age may therefore prove useful in aiding diagnosis andpossibly prognosis of NMS; however further work is required to validatethis possibility. These observations are consistent with the conclusionof a delayed, or interrupted, conversion from intra- to extra-uterinelife in ill, neonatal foals, particularly those with NMS. This mechanismmay be similar to that reported in foals of mares treated with theprogestagen altrenogest which have a slower adaptation to theextra-uterine environment (Neuhauser, et al., (2008) Exp Clin EndocrinolDiabetes 116, 423-8.).

Pregnane profiles did not appear to differ between mild-moderate andseverely affected foals although it is likely that a larger populationneeds to be sampled to detect such differences. Furthermore, thecategorization used may have been inappropriate for finding suchdifferences. In previous studies, pregnane concentrations decreased infoals with NMS as they displayed clinical improvement (Rossdale, et al.,(1995) Reprod Fertil Dev 7, 567-575). This observation could not bevalidated by the current study as concentrations were only measured overthe first 48 h of life. Pregnane concentrations were not significantlydifferent between survivors and non-survivors in this study and, again,the short sampling period is likely to have precluded the ability todetect this finding. The effect of a known hypoxic episode on plasmapregnane profiles was examined due to the suggested aetiological role ofhypoxia in NMS. Hypoxia did not appear to have an effect on pregnaneconcentrations; however it is impossible to accurately evaluate thiscriterion, particularly with regard intra-uterine hypoxia.

Differences in concentrations of pregnenolone and pregnanediol betweensick and healthy foals have previously been described (Rossdale, et al.,(1995) Reprod Fertil Dev 7, 567-575). Pregnanediol was not consistentlymeasureable in the current foal population whereas the androgens,androstenedione and epitestosterone, have not previously been identifiedin foals with NMS (Rossdale, P. D. (2004) In: Proceedings, 51st AmericanAssociation of Equine Practitioners, Denver, Colo. pp 75-126). It islikely that these analytes were simply not investigated in theanalytical method originally developed (Houghton, et al., (1991) JReprod Fertil 44, 609-617). LC-MS allows better differentiation of theindividual steroids than can be achieved by radioimmunoassay (Rossdale,et al., (1997) Equine Vet J 24, S96-S99).

The cause of the increased plasma pregnane concentrations detected inill neonatal foals cannot be elucidated from this study; however theauthors propose that these concentrations occur as a result ofpersistence of foetal signals for the in utero state of being quiet andnon-ambulatory. Certain pregnanes, such as progesterone, and theirmetabolites have neuromodulatory, anaesthetic and anxiolytic propertiesimportant for tonic inhibition of foetal CNS activity and damping foetalmovement to prevent maternal damage (Mellor, et al., (2005) Brain ResRev 49, 455-471). The receptors in the foetal brain are more sensitiveto these pregnanes, compared with the receptors in the adult brain(Crossley, et al., (2000) Neuropharmacology 39, 1514-1522). Infusion ofthe neurosteroid pregnane allopregnanolone to healthy neonatal foalsinduces obtundation, lack of affinity for the mare, and decreasedresponse to external stimuli (Madigan, et al., (2012) Equine Vet J44:S41 109-112). These effects were short-lasting and associated withmeasurable concentrations of pregnanes (Madigan, et al., (2012) EquineVet J 44:S41 109-112). This suggests that these steroids can cross theblood brain barrier and exert neuromodulatory effects, which at highconcentrations may have a dampening effect in the CNS with resultingalterations in states of consciousness, altered behaviour, andresponsiveness to stimuli, such as observed in NMS cases. The oppositemay also apply; when certain pregnane concentrations are low, foals maybe more alert, responsive to environmental stimuli, have affinity forthe dam, and nurse. Specific enzymes may be inhibited in these foals andthe roles of 5α-reductase, 3β-hydroxysteroid dehydrogenase and3α-hydroxysteroid dehydrogenase need to be further evaluated. It hasbeen suggested that the 5α-reduction step may be critical in determiningthe quantity of 5α-reduced pregnane metabolites either produced fromprogesterone within the foetal brain or derived from precursors enteringthe brain from the blood (Nguyen, et al., (2003) Pediatr Res 53,956-964). The underlying cause of any possible abnormal adrenal functionis also not known; it may reflect a state of dysmaturity in which thefoal fails to transition to extra-uterine life or may reflect hypoxicinjury to the HPA axis (Rossdale, et al., (1997) Equine Vet J 24,S96-S99). Another potential reason for persistence of foetal hormones isa failure of normal events of parturition which are an essential part ofthe signaling that the foal has transitioned to outside of the mare andthe desire to rise and nurse can commence. The foetal state of sedationand limited movement in utero is critical to survival of the foal andmare and would require clear unequivocal signals of transition tooutside of the uterus (birth). Events such as shorter times in the birthcanal or altered deliveries might produce a failure of transitionsignals to allow foetal hormone reduction and the transition from the inutero foetal cortical status to extra-uterine behavioural status.Regulation of the neuroactive steroid content in the foetal ovine brainis independent of adrenal steroidogenesis and hypothalamic-pituitaryfactors (Nguyen, et al., (2004) J Endocrinol 182, 81-88) however, in theneonate, concentrations of some neurosteroids and their precursors inthe peripheral circulation dramatically affect concentrations in thebrain (Nguyen, et al., (2003) Pediatr Res 53, 956-964). Lastly, anotherpossible mechanism would be the reversion to foetal cortical status whenadverse post birth circumstances occur. The syndrome of reversion tofoetal circulation is a well-known and accepted consequence of adversebirth and post birth events which cause the neonate to revert tomechanisms which regulated the cardiovascular system in utero.

It is also possible that the elevated pregnanes are acting in aneuroprotective role as has been reported in other species. Stress(hypoxia, endotoxin) in the neonatal period increases neurosteroidconcentrations in the brain of newborn lambs (Hirst, et al., (2006)Neuroendocrinol 84, 264-274; Billiards, et al., (2002) Pediatr Res 52,892-899), which is suggested to represent an endogenous protectivemechanism. Similarly, acute, but not chronic, hypoxic stress duringpregnancy increases foetal neurosteroid concentrations (Hirst, et al.,(2006) Neuroendocrinol 84, 264-274). Indeed inhibition of neurosteroidsynthesis increases asphyxia-induced brain injury in late gestationfoetal sheep (Yawno, et al., (2007) Neurosci. 146, 1726-33).

Phenotypical characteristics of “maladjusted foals” may have more thanone etiology (hypoxic/ischemic versus non-hypoxic/ischemic). Thenon-hypoxic foal is the one that lacks the normal transition fromsynthesis to inhibition of specific neurosteroids for readiness forbirth (from foetal to neonatal neurosteroid profile). This may explainwhy some affected foals have a relatively fast recovery with noremaining long-term neurological deficits, and no apparent or knownhypoxic events prior, during or shortly after birth.

Plasma concentrations of progestagens were measured in this study butideally concentrations in brain tissue, which are known to be muchhigher than those in the peripheral circulation, would be measured.Neurosteroids and their precursors are known to cross the blood brainbarrier (Wang, et al., (1997) J Steroid Biochem Mol Biol. 62, 299-306)and are extremely potent such that small concentrations can have largelocal effects in neuronal tissue. Further, many steroids are metabolizedto other compounds prior to exerting their effects.

In conclusion, specific alterations in pregnane profiles were detectedbetween healthy control foals and ill, neonatal foals presenting toNICU. The anaesthetic and sedative properties of these pregnanes mayaccount for the behavioural alterations seen in maladjusted and illfoals. These differences may reflect a delayed or interrupted transitionfrom foetal to neonatal hypothalamic pituitary adrenocortical status.Repeated measurements of these pregnanes over time may be useful fordistinguishing between foals with NMS and other neonatal disorders.Increased pregnane concentrations may cross the blood brain barrier andbe responsible for some of the behavioural and neurological alterationsobserved in foals with NMS.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of identifying and/or diagnosing asyndrome of reversion to fetal consciousness in a neonate mammalcomprising: a) measuring the pregnane levels in a biological sample fromthe neonate mammal and; b) identifying and/or diagnosing the neonatemammal as suffering reversion to fetal consciousness when elevatedpregnane levels are measured.
 2. A method of identifying and/ordiagnosing a syndrome of reversion to fetal consciousness in a neonatemammal comprising: a) receiving a report providing the pregnane levelsin a biological sample from the neonate mammal and; b) identifyingand/or diagnosing the neonate mammal as suffering reversion to fetalconsciousness when elevated pregnane levels are measured.
 3. The methodof any one of claims 1 to 2, wherein the neonate mammal is an equine. 4.The method of any one of claims 1 to 3, wherein the neonate mammal isexhibiting symptoms of reversion to fetal consciousness.
 5. The methodof any one of claims 1 to 4, wherein the pregnanes measured compriseneurosteroidal activity.
 6. The method of any one of claims 1 to 5,wherein one or more pregnanes selected from the group consisting ofprogesterone, pregnenolone, 17OH-progesterone and pregnanediol aremeasured.
 7. The method of any one of claims 1 to 6, wherein one or morepregnanes selected from the group consisting of progesterone andpregnenolone are measured.
 8. The method of any one of claims 6 to 7,wherein the elevated pregnenolone levels are above about 6000 ng/mlunder 24 hours post birth, above about 550 ng/ml at 24-28 hours postbirth and/or above about 315 ng/ml at or after 48 hours post birth. 9.The method of any one of claims 6 to 8, wherein the elevatedpregnenolone levels are above about 12,900 ng/ml under 24 hours postbirth, above about 3470 ng/ml at 24-28 hours post birth and/or aboveabout 3420 ng/ml at or after 48 hours post birth.
 10. The method of anyone of claims 6 to 9, wherein the elevated progesterone levels are aboveabout 10.5 ng/ml under 24 hours post birth, above about 0.8 ng/ml at24-28 hours post birth and/or above about 0.1 ng/ml at or after 48 hourspost birth.
 11. The method of any one of claims 6 to 10, wherein theelevated progesterone levels are above about 9.7 ng/ml at 24-28 hourspost birth and/or above about 14.3 ng/ml at or after 48 hours postbirth.
 12. The method of any one of claims 1 to 11, further comprisingmeasuring androgen levels in the biological sample.
 13. The method ofclaim 12, wherein one or more androgens are selected from the groupconsisting of androstenedione, dehydroepiandrosterone (DHEA),DHEA-sulphate and epitestosterone are measured.
 14. The method of anyone of claims 1 to 13, wherein the biological sample is selected fromthe group consisting of blood, serum, plasma and urine.
 15. The methodof any one of claims 1 to 14, wherein the neonate is less than 120 hourspost birth.
 16. The method of any one of claims 1 to 15, wherein thepregnane levels are measured at two or more time points.
 17. The methodof any one of claims 1 to 16, wherein upon measuring elevated pregnanelevels and positively diagnosing the neonate mammal as sufferingreversion to fetal consciousness, administering to the neonate aneffective amount of a 5α-reductase inhibitor.
 18. The method of claim17, wherein the 5α-reductase inhibitor is selected from the groupconsisting of alfatradiol, dutasteride, finasteride, bexlosteride,epristeride, izonsteride, lapisteride, turosteride, and analogs, saltsand mixtures thereof.
 19. The method of any one of claims 1 to 15,wherein upon measuring elevated pregnane levels and positivelydiagnosing the neonate mammal as suffering reversion to fetalconsciousness, subjecting the neonate to squeezing along the mid-thorax.20. The method of claim 19, wherein the applied pressure of thesqueezing is maintained for at least 20 minutes.
 21. A method ofreducing, ameliorating, mitigating and/or reversing a syndrome ofreversion to fetal consciousness in a neonate mammal comprisingadministering to the neonate an effective amount of a 5α-reductaseinhibitor.
 22. The method of claim 22, wherein the 5α reductaseinhibitor is selected from the group consisting of alfatradiol,dutasteride, finasteride, bexlosteride, epristeride, izonsteride,lapisteride, turosteride, and analogs, salts and mixtures thereof.
 23. Amethod of reducing, ameliorating, mitigating and/or reversing a syndromeof reversion to fetal consciousness in a neonate mammal comprisingsubjecting the neonate to squeezing along the mid-thorax.
 24. The methodof claim 23, wherein the applied pressure of the squeezing is maintainedfor at least 20 minutes.
 25. The method of any one of claims 21 to 24,wherein the neonate mammal has elevated levels of pregnanes in itsblood, serum, plasma and/or urine.
 26. The method of any one of claims21 to 25, wherein one or more pregnanes selected from the groupconsisting of progesterone, pregnenolone, 17OH-progesterone andpregnanediol are measured.
 27. The method of any one of claims 21 to 26,wherein one or more pregnanes selected from the group consisting ofprogesterone and pregnenolone are measured.
 28. The method of any one ofclaims 26 to 27, wherein the elevated pregnenolone levels are aboveabout 6000 ng/ml under 24 hours post birth, above about 550 ng/ml at24-28 hours post birth and/or above about 315 ng/ml at or after 48 hourspost birth.
 29. The method of any one of claims 26 to 28, wherein theelevated pregnenolone levels are above about 12,900 ng/ml under 24 hourspost birth, above about 3470 ng/ml at 24-28 hours post birth and/orabove about 3420 ng/ml at or after 48 hours post birth.
 30. The methodof any one of claims 26 to 29, wherein the elevated progesterone levelsare above about 10.5 ng/ml under 24 hours post birth, above about 0.8ng/ml at 24-28 hours post birth and/or above about 0.1 ng/ml at or after48 hours post birth.
 31. The method of any one of claims 26 to 30,wherein the elevated progesterone levels are above about 9.7 ng/ml at24-28 hours post birth and/or above about 14.3 ng/ml at or after 48hours post birth.
 32. An inflatable cuff for squeezing the mid-thorax ofa neonate mammal comprising a pneumatic inflating sleeve of sufficientcircumference to encircle the mid-thorax of a neonate mammal.
 33. Thecuff of claim 32, wherein the pneumatic inflating sleeve is of asufficient circumference to encircle the mid-thorax of an equineneonate.
 34. The cuff of any one of claims 32 to 33, wherein, wheninflated, the sleeve of the cuff applies substantially even pressurealong the length of the mid-throrax encircled by the sleeve.
 35. Thecuff of any one of claims 32 to 34, wherein the cuff further comprisesstraps for stably positioning the cuff around the mid-thorax.
 36. Thecuff of claim 35, wherein the straps are configured to encircle the neckof the mammal.
 37. The cuff of any one of claims 32 to 36, in fluidcommunication with a foot pump or motorized pump capable of and suitablefor inflating the cuff.
 38. The cuff of any one of claims 32 to 37,wherein the cuff is inflatable to about 10 psi.
 39. A device comprisingtwo inflatable cuffs of any one of claims 32 to 38.